WO2005059178A1

WO2005059178A1 - Improved selective ligation and amplification assay

Info

Publication number: WO2005059178A1
Application number: PCT/US2004/041480
Authority: WO
Inventors: Tom Morrison
Original assignee: Bio Trove, Inc.
Priority date: 2003-12-10
Filing date: 2004-12-10
Publication date: 2005-06-30
Also published as: JP2007515956A; EP1692314A1; CA2549849A1

Abstract

An improved assay for identifying and distinguishing one or more a single nucleotide polymorphisms in one or more target sequences of nucleic acid comprises, in a single-tube reaction system, three or more primers, two of which bind to a target nucleic acid sequence, flanking a SNP, so that the 3'-end of one or more first primers is adjacent to the 5'-end of a second primer, the two primers being selectively ligated and then amplified by a third primer to exponentially produce the complementary strand of the one or more target sequences. The other strand of the one or more target sequences are exponentially amplified by one or more hybridizable probes, each labeled with a different fluorophore, the fluorophore-labeled hybridizable probes being quenched until incorporation into and amplification of target nucleic acid products. Also provided is a method for identifying one or more SNPs in one or more target sequences of nucleic acid in each single through-hole of a nanoliter sampling array, and a kit for such a method containing a nanoliter sampling array chip, primer sequences, and reagents required to selectively ligate primers for amplification of desired target nucleic acid sequences.

Description

Improved Selective Ligation and Amplification Assay

Technical Field The present invention relates to assays for amplifying and identifying target sequences of nucleic acids involving a combined ligation and amplification protocol, and the use of nanoliter sampling arrays to perform such assays.

Background Art Genetic variations are increasingly being linked to a multitude of disease conditions and predispositions for disease, including cancer, multiple sclerosis, autoimmune diseases, cystic fibrosis, and schizophrenia. The ability to identify genetic variations rapidly and inexpensively will greatly facilitate diagnosis, risk assessment, and determination of the prognosis for such diseases and predispositions for these diseases. One possibility for identifying genetic variations involves combining selective ligation and amplification techniques, disclosed in U.S. Patent No. 5,593,840 to

Bhatnagar et al. and U.S. Patent No. 6,245,505 to Todd et al, both of which are hereby incorporated by reference herein. Both patents disclose the use of at least three primers, two of which are complementary to adjacent regions of the 3 '-end of one strand of a target nucleic acid sequence which, after hybridization, can be ligated and then extended. In Todd et al, the third primer is a random sequence, complementary to the random sequence at the 3 '-end of the downstream primer (that ligates to the upstream primer) and identical to the random sequence on the 5'-end of the first primer. In Bhatnagar et al., the third primer is complementary to the upstream primer, and also to the opposite strand of the target sequence. In both cases, there must be complementarity at the 3'-end of the third primer to allow amplification to occur. A heat-stable polymerase is used to amplify the target nucleic acid sequence, and both the ligation and amplification reactions can be carried out in the same reaction mixture. An optional gap between the adjacent primers may be present, which may be filled by a polymerase to allow successful ligation of the adjacent primers. Such a system allows identification of genetic variability in target nucleic acid sequences, and identification of multiple alleles. Summary of the Invention In a first embodiment of the invention, there is provided an improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, the assay being a selective ligation and amplification method of the type using a controlled-temperature reaction mixture including the target sequence, ligatable first and second primers having at least a portion substantially complementary to first and second segments of the target sequence, respectively, and a third primer that is substantially complementary to a random sequence segment of the first and second primers, wherein the improvement comprises: distinguishing in a single-tube reaction system between one or more SNPs in one or more target sequences of nucleic acid using two unique probes designed to hybridize to the target nucleic acid sequences with SNPs of interest, each hybridizable probe having a different fluorescent tag that is quenched until incorporation of the probe into amplified target nucleic acid product. In some embodiments of the improved assay, of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises one or more SNPs of interest that are not at an end of the target sequence, the assay being a selective ligation and amplification method of the type using a thermocycled reaction mixture including the target sequence, a first primer having at least a portion of its 3 '-end substantially complementary to a first segment at a first end of the target sequence, a second primer having at least a portion of its 5'-end substantially complementary to a second segment at a second end of the target sequence, the 5'-end of the second primer being adjacent to or within two to four bases of the 3 '-end of the first primer wherein a nucleotide complementary to the SNP of the target sequence is present at either the 3 '-end of the first primer or at the 5 '-end of the second primer, and a third primer that is substantially complementary to a random sequence segment at the 3'-end of the second primer and to a substantially similar sequence at the 5 '-end of the first primer, at least four different nucleotide bases, a thermostable polymerase and a thermostable ligase, wherein the improvement comprises distinguishing in a single-tube reaction system between one or more SNPs in one or more target sequences of nucleic acid using two unique probes designed to hybridize to the target nucleic acid sequences with SNPs of interest, each hybridizable probe having a different fluorescent tag that is quenched until incorporation of the probe into amplified target nucleic acid product. The first hybridizable probe with first fluorescent tag has a unique random sequence that hybridizes to a first amplified target nucleic acid generated by the third primer from a ligated first primer-second primer product having a first SNP of interest on the 3 '-end of the first primer, the first hybridizable probe thereby becoming incorporated into amplified opposite strand target nucleic acid product to give a first fluorescent signal. The second hybridizable probe with second fluorescent tag has a unique random sequence that hybridizes to a second amplified product generated by the third primer from a different ligated first primer- second primer product having a second SNP of interest on the 3'-end of the first primer, the second hybridizable probe thereby becoming incorporated into amplified opposite strand target nucleic acid product to give a second fluorescent signal. In a preferred embodiment, the random sequences of the first and second hybridizable probes are unique sequences, such that specific incorporation of each of the hybridizable probes into amplified target nucleic acid preferentially occurs after ligation of the first primer-second primer product having the particular SNP of interest that the hybridizable probe was designed to detect. Upon incorporation of the hybridizable probe into amplified product, fluorescence occurs, making detection of the amplified product distinguishable from non-specific background products. Additionally, the random sequence of the third primer is also a unique sequence, optimized for PCR to reduce non- specific amplified products that may be generated in the presence₍ of human or other species chromosomes to a sufficiently low level that such non-specific products do not interfere with detection of amplified products having a SNP of interest. Alternatively, the two hybridizable probes do not contain fluorescent tags, but are simply additional primers designed to distinguish different ligated products having different SNPs of interest. Detection of amplified product with a SNP of interest is then done using additional hybridizable probes, similar to the additional primers, but are developed in a manner not to interfere with amplification. These hybridizable probes have a fluorescent tag, or alternatively, each have a different fluorescent tag, and upon hybridizing to amplified product, fluoresce, thereby allowing detection of amplified product. In another embodiment of the invention there is provided an improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises a SNP of interest that is not at an end of the target sequence, the assay being a selective ligation and amplification method of the type using a thermocycled reaction mixture including the target sequence, a first primer having at least a portion of its 3 '-end substantially complementary to a first segment at a first end of the target sequence, a second primer having at least a portion of its 5 '-end substantially complementary to a second segment at a second end of the target sequence, the 5 '-end of the second primer being adjacent to the 3 '-end of the first primer wherein a nucleotide complementary to the SNP of the target sequence is present at either the 3 '-end of the first primer or at the 5 '-end of the second primer, and a third primer that is substantially complementary to a random sequence segment at the 3 '-end of the second primer and to a substantially similar sequence at the 5 '-end of the first primer, at least four different nucleotide bases, a thermostable polymerase and a thermostable ligase, wherein the improvement comprises homogeneously detecting amplified target sequence using a dye specific for binding to double-stranded (ds) DNA that fluoresces upon binding target sequence. In a preferred embodiment, the random sequence of the third primer is a unique sequence, optimized for PCR such that no non-specific products are generated in the presence of human or other species chromosomes. In some embodiments, primers may be affixed on, within or under a biocompatible material such as a wax-like coating on the surface of the through-holes by drying the primers after application to the through- holes, wherein the biocompatible material may comprise, for example, a polyethylene glycol (PEG) material. Alternatively, assays in accordance with the present invention may use a thermostable polymerase that lacks 5' to 3' exonuclease activity, or a thermostable polymerase that lacks 3' to 5' exonuclease activity, or a thermostable polymerase that lacks both 5' to 3' and 3' to 5' exonuclease activity. Examples of thermostable polymerases which lack 5' to 3' exonuclease activity include Stoffel fragment, Isis™ DNA polymerase, Pyra™ exo(-) DNA polymerase, and Q-BioTaq™ DNA polymerase. Examples of thermostable polymerases which lack 3' to 5' exonuclease activity include Taq polymerase, SurePrime™ Polymerase, and Q-BioTaq™ DNA polymerase. An example of a thermostable polymerase which lacks both 5' to 3' and 3' to 5' exonuclease activity is Q-BioTaq™ DNA polymerase. Suitable dyes include SYBR^® Green I and SYBR^® Green π, YOYO^®-l , TOTO^®-l , POPO^®-3, ethidium bromide, or any other dye that allows rapid, sensitive detection of amplified target nucleic acid sequence using fluorescence. In another embodiment, there is provided a nanoliter sampling array comprising a first platen having at least one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes. In this particular embodiment, each through-hole contains at least a first primer having at least a portion of its 3 '-end substantially complementary to a first segment at a first end of a potential nucleic acid target sequence a second primer having at least a portion of its 5 '-end substantially complementary to a second segment at a second end of the potential nucleic acid target sequence, the 5 '-end of the second primer being adjacent to the 3 '-end of the first primer upon binding to the potential nucleic acid target sequence. In addition, the sampling array may further comprise a second platen having at least one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes wherein the first and second platen are fixedly coupled such that the through-holes of each are aligned. In yet another embodiment, there is provided a method of identifying a SNP in a target sequence of nucleic acid, the method comprising providing a first sample platen having a high-density microfluidic array of through-holes, each through-hole having a first primer having at least a portion of its 3'-end substantially complementary to a first segment at a first end of the target sequence, a second primer having at least a portion of its 5'-end substantially complementary to a second segment at a second end of the target sequence, the 5 '-end of the second primer being adjacent to the 3 '-end of the first primer, and third primer that is substantially complementary to a random sequence segment at the 3 '-end of the second primer and to the 5 '-end of the first primer, introducing a sample containing a target sequence of nucleic acid having a SNP of interest to the array, introducing reagents to the through-holes in the array, the reagents including a thermostable polymerase, a thermostable ligase, and at least four different nucleotide bases, thermocycling the array, and detecting amplified target sequence. In a preferred embodiment, primers 1 and 2 are designed with a possible match to the target strand SNP located at either the 3 '-end of the 5' primer (the first primer) or located at the 5 '-end of the 3' primer (the second primer). When the first and second primers hybridize to the target strand, adjacent to each other and flanking the SNP, ligation of the primers only occurs if there is a successful match to the SNP by one of the primers. In this way, the ligation is selective and so selective amplification of the desired target sequence containing the SNP of interest also occurs. As described above, in some embodiments, primers may be affixed on, within or under a biocompatible material such as a wax-like coating on the surface of the through-holes by drying the primers after application to the through-holes, wherein the biocompatible material may comprise, for example, a polyethylene glycol (PEG) material. In addition, the method of identifying a SNP in a target sequence of nucleic acid may additionally comprise using a thermostable polymerase that lacks 5' to 3' exonuclease activity, and detecting amplified target sequence using a dye specific for binding to double-stranded (ds) DNA that fluoresces upon binding target sequence.

Alternatively, detecting may comprise using first primers and second primers designed to generate amplified target sequences with differential melting curves to distinguish individual amplified target sequences by differences in melting temperatures (T_ms), or may comprise using a probe specific for hybridizing across a ligation junction formed between the first primer and second primer after binding to the target sequence wherein the probe specific for hybridizing across the ligation junction has a fluorescent group and a fluorescence-modifying group, or using a probe containing a fluorescent group and a fluorescence-modifying group specific for hybridizing to a region of the target sequence wherein upon extension of the probe, the fluorescence-modifying group is excised and the fluorescent group fluoresces. Additionally, detection may be done using a probe specific for hybridizing to any unique sequence in the amplified target nucleic acid, the probe having a fluorescent group and a fluorescence-modifying group such that the upon hybridization the probe fluoresces, allowing detection of the amplified target nucleic acid. Other means of detection comprise the use of amplification primers which match the random sequence of primer 2 wherein the primers are labeled with a fluorescent group that only fluoresces when incorporated in a PCR product, similar to Lux™ primers known in the art. In such an embodiment, the fluorescent group is quenched by secondary structure before incorporation into double-stranded product, such that prior to incorporation, a sequence in the primer/probe binds to a complementary sequence in the primer/probe containing the fluorescent group, quenching the fluorescent group. In another embodiment, primers 1 and 2 are Fluorescence Resonance Energy Transfter (FRET) partners, such that when hybridized to the amplified target sequence, produced only after primers 1 and 2 are ligated and amplified, they fluoresce. Yet another embodiment provides a kit for use in identification of amplified target nucleic acid sequences, the kit comprising a sample platen having one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes. In the array of the kit, each through-hole contains at least a first primer having at least a portion of its 3 '-end substantially complementary to a first segment at a first end of potential nucleic acid target sequence, and a second primer having at least a portion of its 5 '-end substantially complementary to a second segment at a second end of the potential nucleic acid target sequence, the 5'-end of the second primer being adjacent to the 3'-end of the first primer upon binding to the potential nucleic acid target sequence. The kit also comprises a reagent platen having a high-density microfluidic array of through-holes, each through-hole containing a third primer that is substantially complementary to a random sequence segment at the 3 '-end of the second primer and to a substantially similar sequence at the 5'-end of the first primer, at least four different nucleotide bases, a thermostable polymerase, and a thermostable ligase. In the kit of this embodiment, the reagent platen has a structural geometry that corresponds to the sample platen, thereby allowing delivery of reagent components and target nucleic acid sample to the primers in the sample platen. In other embodiments, the thermostable polymerase may lack 5' to 3' exonuclease activity.

Brief Description of the Drawings The foregoing features of the invention will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which: Fig. 1-A, shows a double-stranded target nucleic acid sequence with a single nucleotide polymorphism (SNP). Fig. 1-B1 shows a denatured 3' to 5' target strand with primers 1 and 2 hybridized adjacent to the SNP, the base complementary to the SNP located at the 3 '-end of primer 1 and shows the random sequence (RS) of primer 3 hybridized to 3'-end of the ligated Pl- P2 product. Fig. 1-B2 shows a denatured 3' to 5' target strand with primers 1 and 2 hybridized adjacent to the SNP, the base complementary to the SNP located at the 5 '-end of primer 2 and shows the random sequence (RS) of primer 3 hybridized to 3'-end of the ligated Pl- P2 product. Fig. 1-C shows a denatured 5 '-3' target nucleic acid strand being extended by un- ligated primer PI. Fig. 2-A shows a double-stranded target nucleic acid sequence with a single nucleotide polymorphism (SNP). Fig. 2-B shows primers PI and P2 hybridized to a denatured target strand of nucleic acid (the 3' to 5' strand) wherein a base complementary to the SNP in the target strand is present on the 3'-end of PI, and each of primers PI and P2 contain a random sequence at their 5 '-end and 3 '-end, respectively. Fig. 2-C shows ligated P1-P2 product being amplified by primer P3 to produce P3-amplified product. Fig. 2-D shows P3-amplified product being amplified by primer P3 to produce

P3-ampflied product (3' to 5'). Figs. 2-E1 and 2-E2 show exponential amplification of P3-amplified product (5' to 3') and P3-amplified product (3' to 5'), respectively. Fig. 3 shows a cartoon of the dye SYBR Green I binding to double-stranded amplified target nucleic acid and fluorescing. Fig. 4-A shows upstream primer A-B, downstream primer C-D, and general extension primer D' with a target nucleic acid having a SNP of interest in a single-tube reaction system for distinguishing between one or more SNPs in one or more target sequences of nucleic acid, the single-tube reaction system also containing upstream primer F-E and a second nucleic acid target with a second SNP of interest. Fig. 4-B shows ligation of upstream primer A-B with downstream primer C-D when successful match-up occurs with a first SNP of interest in a first target sequence of nucleic acid, and also shows ligation of upstream primer F-E with down stream primer C- D when successful match-up occurs with a second SNP of interest in a second target sequence of nucleic acid present in the same tube. Fig. 4-C shows extension of ligation products A-B-C-D and F-E-C-D by general extension primer D'. Fig. 4-D shows hybridization of hybridizable probe A with fluorescent tag 1 to extended product A'-B'-C'-D' and hybridization of hybridizable probe F with fluorescent tag 2 to extended product F'-E'-C'-D'. Fig. 4-E shows incorporation and amplification of a first target nucleic acid with a first SNP of interest by hybridizable probe A, triggering fluorescence of fluorophore 1 in a first amplified product, and incorporation and amplification of a second target nucleic acid with a second SNP of interest by hybridizable probe F, triggering fluorescence of fluorophore 2 in a second amplified product. Fig. 5A shows upstream primer A-B, downstream primer C-D, and general extension primer D' with a target nucleic acid having a SNP of interest in a single-tube reaction system for distinguishing between one or more SNPs in one or more target sequences of nucleic acid , the single-tube reaction system also containing upstream primer F-E and a second nucleic acid target with a second SNP of interest in an alternative embodiment of the single-tube reaction system of Fig. 4. Fig. 5B shows ligation of upstream primer A-B with downstream primer C-D when successful match-up occurs with a first SNP of interest in a first target sequence of nucleic acid, and also shows ligation of upstream primer F-E with down stream primer C-

D when successful match-up occurs with a second SNP of interest in a second target sequence of nucleic acid present in the same tube. Fig. 5C shows extension of ligation products A-B-C-D and F-E-C-D by general extension primer D'. Fig. 5D shows hybridization of primer A with no fluorescent tag to extended product A'-B'-C'-D' and hybridization of primer F with no fluorescent tag to extended product F'-E'-C'-D'. Fig. 5E shows amplification of a first target nucleic acid with a first SNP of interest by primer A to produce a first amplified product, and amplification of a second target nucleic acid with a second SNP of interest by primer F, to produce a second amplified product. Fig. 5F shows a competing reaction to the amplification reactions in Fig. 5E, wherein incorporation and low-efficiency production of a first target nucleic acid with a first SNP of interest is carried out by hybridizable probe A, triggering fluorescence of fluorophore 1 in a first product, thereby allowing detection of a first amplified target nucleic acid, and wherein incorporation and low-efficiency production of a second target nucleic acid with a second SNP of interest is carried out by hybridizable probe F, triggering fluorescence of fluorophore 2 in a second product, thereby allowing simultaneous detection of a second amplified target nucleic acid. Fig. 6 shows a typical high-density sample array of through-holes according to the prior art.

Detailed Description of Specific Embodiments Definitions. As used in this description and the accompanying claims, the following terms shall have the meanings indicated, unless the context otherwise requires: "Target nucleic acid," "target nucleic acid sequence" or "potential target nucleic acid sequence" means any prokaryotic or eukaryotic DNA or RNA including from plants, animals, insects, microorganisms, etc. It may be isolated or present in samples which contain nucleic acid sequences in addition to the target nucleic acid sequence to be amplified. The target nucleic acid sequence may be located within a nucleic acid sequence which is longer than that of the target sequence. The target nucleic acid sequence may be obtained synthetically, or enzymatically, or can be isolated from any organism by methods well known in the art. Particularly useful sources of nucleic acid are derived from tissues or blood samples of an organism, nucleic acids present in self- replicating vectors, and nucleic acids derived from viruses and pathogenic organisms such as bacteria and fungi. Also particularly useful are target nucleic acid sequences which are related to disease states, such as those caused by chromosomal rearrangement, insertion, deletion, translocation and other mutation, those caused by oncogenes, and those associated with cancer. "Selected" means that a target nucleic acid sequence having the desired characteristics is located and probes are constructed around appropriate segments of the target sequence. "Probe" or "primer" has the same meaning herein, namely, a nucleic acid oligonucleotide sequence which is single-stranded. The term ohgonucleotide includes DNA, RNA and PNA. A probe or primer is "substantially complementary" to the target nucleic acid sequence if it hybridizes to the sequence under renaturation conditions so as to allow target-dependent ligation or extension. Renaturation depends on specific base pairing between A-X (where X is T or U) and G-C bases to form a double-stranded duplex structure. Therefore, the primer sequences need not reflect the exact sequence of the target nucleic acid sequence. However, if an exact copy of the target sequence is desired, the primer should reflect the exact sequence. Typically, a "substantially complementary" primer will contain at least 70% or more bases which are complementary to the target nucleic acid sequence. More preferably 80% or the bases are complementary, and still more preferably more than 90% of the bases are complementary. Generally, the primer should hybridize to the target nucleic acid sequence at the end to be ligated or extended to allow target-dependent ligation or extension. Primers may be RNA or DNA and may contain modified nitrogenous bases which are analogs of the normally incorporated bases, or which have been modified by attaching labels or linker arms suitable for attaching labels. Inosine may be used at positions where the target sequence is not known, or where it may be degenerate. The oligonucleotides should be sufficiently long to allow hybridization of the primer to the target sequence and to allow amplification to proceed. They are preferably 15 to 50 nucleotides long, more preferably 20-40 nucleotides long, and still more preferably 25-35 nucleotides longs. The nucleotide sequence of the primers, both content and length, will vary depending on the target sequence to be amplified. It is contemplated that a primer may comprise one or more oligonucleotides which comprise substantially complementary sequences to the target nucleic acid sequence. Thus, under less stringent conditions, each of the oligonucleotide primers would hybridize to the same segment of the target sequence. However, under increasingly stringent conditions, only that oligonucleotide primer which is most complementary to the target nucleic acid sequence will hybridize. The stringency of the hybridization conditions is generally known to those in the art to be dependent on temperature, solvent, ionic strength, and other parameters. One of the most easily controlled parameters is temperature and since conditions for selective ligation and amplification are similar to those for PCR reactions, one skilled in the art can determine the appropriate conditions required to achieve the level of stringency desired. Primers suitable for use in the present invention may be derived from any method known in the art, including chemical or enzymatic synthesis, or by cleavage of larger nucleic acids using non-specific nucleic acid-cleaving chemicals or enzymes, or by using site-specific restriction endonucleases. In order for the ligase of the present invention to ligate the primers together, the primers used are preferably phosphorylated at their 5'-ends. This may be achieved by any known method in the art, including use of T4 polynucleotide kinase. The primers may be phosphorylated in the presence of unlabeled or radiolabeled ATP. The term "four different nucleotide bases" means deoxythymidine triphosphate (dTTP), deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP) when the context is DNS, and means uridine triphosphate (UTP), adenosine triphosphate (ATP), cytidine triphosphate (CTP), and guanosine triphosphate (GTP) when the context is RNA. Alternatively, dUTP, dITP (deoxyinosine triphosphate), rITP (riboinosine triphosphate) or any other modified base may replace any one of the four nucleotide bases or may be included along with the four nucleotide bases in the reaction mixture so as to be incorporated into the amplified strand.

The amplification steps are conducted in the presence of at least the four deoxynucleoside triphosphates (dATP, dCTP, dGTP and dTTP) or a modified nucleoside triphosphate to produce a DNA strand, or in the present of the four ribonucleoside triphosphates (ATP, CTP, DTP and UTPO or a modified ribonucleoside triphosphate to produce an RNA strand from extension of the primer. The term "adequate detection of desired amplified product" means detection of at least a two-fold increase in desired amplified target strand over competing linear products. The term "target sequence detectable above linearly amplified product" means that target sequence is amplified at least two-fold over that of competing linearly amplified non-ligated primer product. The term "random sequence" as used herein means a sequence unrelated to the target sequence or chosen not to bind to the target sequence or other sequences that might be expected to be present in a test sample. The term "biocompatible material" as used herein means that the material does not prevent biological processes, such as enzymatic reactions, from occurring when the biocompatible material is present, does not eliminate biological activity or required secondary, tertiary or quaternary structure of biomolecules, such as nucleic acids and proteins, and in general, is not incompatible with biological processes and molecules. The term "first and second primers being ligatable upon binding to the nucleic acid target sequence" as used herein, means that the first and second primers bind potential target nucleic acid with the 3'-end of the first primer adjacent to, or within about a one- to four- nucleotide gap of, the 5 '-end of the second primer, such that subjecting the hybridized first and second primers to appropriate enzymatic or non-enzymatic ligation conditions, including optionally adding a polymerase activity to fill in the gap, allows the first and second primers to be enzymatically or non-enzymatically ligated into a single ligated nucleic acid product. The term "polymerase" as used herein, means any oligomer synthesizing enzyme, including polymerases, helicases, and other protein fragments capable of polymerizing the synthesis of oligomers. The term "controlled-temperature reaction mixture" as used herein means, any reaction mixture wherein temperature is controlled by means of a thermocycle apparatus, an isothermal apparatus, or any other means known to allow temperature control of a reaction, including temperature-controllable environments such as water, oil and sand baths, incubation chambers, etc. The general assay for identifying single-nucleotide polymorphisms (SNPs) that are not at an end of a target sequence through detection of amplified target sequences, using a dye specific for binding to double-stranded DNA that fluoresces upon binding target sequence according to the present invention, is described below and illustrated in Figs. 1-5. The assay can be performed in a single-reaction chamber or container, in a series of reaction chambers or containers, in a nanoliter sampling array having a high- density microfluidic array of hydrophilic through-holes, or in a kit comprising such an array plus necessary reagents. Detection may be homogeneous, and may employ a polymerase that lacks 5' to 3' exonuclease activity, or a polymerase that lacks 3' to 5' exonuclease activity, or a polymerase that lacks both exonuclease activities. The assay can be done with three (PI, P2, P3) or more (A-B, C-D, F-E, D') primers, and is able to detect one or more SNPs in a single target simultaneously. In some versions of the assay, the nucleotide complementary to the SNP of the target nucleotide is present at or near the 5'-end of the second primer P2. In other versions, the nucleotide complementary to the SNP of the target nucleotide is present at or near the 3'- end of the first primer PI . In other versions, there are more than one first primers and second primers, these first and second primers designed to generate amplified target sequences having different melting temperatures, such that the assay is able to distinguish individual amplified target sequences because of their individual, and distinct, T_ms. Assays may be done with first and second primers that contain degenerate base- pairing positions which allow hybridization of variable regions in target sequences adjacent to the SNP, in this way expanding the general flexibility and utility of the assay. Primers 1 and 2, corresponding to 5' and 3' ligation primers, respectively, may be fully or partially complementary to the target sequence. Primer 3 is a generic primer complementary to a random sequence (RS) located at the ends of primers 1 and/or 2 (see Figs. 1 and 2). The 3' end of primer 1 and the 5' end of primer 2 can hybridize either immediately adjacent to each other on the target sequence or can hybridize on the target sequence with a separation, or gap, or one or more nucleotides between them (see Figs. 1- 2 and 4-5). Primers 1 or 2 contain a variant base at or near the 3' end (PI) or the 5' end (P2) to enable the primers to bind to SNPS in a target sequence (see Figs. 1-2). There is also a 3'-hydroxyl group on P2, to facilitate enzymatic or non-enzymatic ligation between PI and P2 or polymerase extension prior to ligation (to fill in any gap). In addition, the

5'-end of P2 can be modified to prevent undesirable ligation to fragments other than PI. Similarly, the 5'-end of PI is phosphorylated to facilitate ligation with P2, and the

3' end of PI may be modified to prevent ligation to fragments other than P2.

Amplification of target nucleic acid is illustrated in Figs. 1 and 2. Temperature is used to denature and anneal target nucleic acid and primers, as required, to allow selective extension of ligation of primers PI and P2. Detection of single-stranded ligation product is carried out using several strategies, some employing a dye specific for binding to double-stranded DNA that is generated either using hybridization probes which hybridize to single-stranded amplified product, or generated after extension and amplification of both the sense and non-sense strands of the ligation product. Other detection strategies employ molecular beacons attached to hybridizable probes. And still other detection strategies employ the use of FRET pairs on hybridizable probes. In some assays, the fluorescent dye is merely added to the reaction mixture, and change in fluorescence intensity is monitored to detect ligated product. In other assays, hybridizable probes are added after generation of ligation product which are specific to the ligation product, and which also contain a molecular beacon, or a fluorescent group and a fluorescence-modifying group. The hybridizable probe may bind to extended ligation product, remaining quenched by the fluorescence- modifying group until extended into amplified product, whereupon the fluorescent group fluoresces and amplified target sequence is detected (see Fig. 4), or the hybridizable probe may be specific for hybridizing across the ligation junction, wherein the probe is again quenched until after hybridizing (see Fig. 5). In the assay illustrated in Fig. 4, one or more hybridizable probes may be used, each having a distinct fluorophore and unique sequence that hybridizes to and amplifies each of one or more target nucleic acid sequences, thereby allowing multiple SNPs to be detected in a single-tube reaction system. Any of the assays may also be carried out in a nanoliter sampling array. The nanoliter array may comprise one or more platens having at least one hydrophobic surface and a high-density microfluidic array of hydrophilic through-holes. The inner surfaces of the through-holes may be coated with a biocompatible material such as a waxlike polyethylene glycol material, or other biocompatible material. Primers may be applied into the through-holes and then dried, either before or after application of the biocompatible material coating, thereby affixing the primers on, within or under the biocompatible material. Target nucleic acids and reagents for processes used in the selective ligation and amplification assay can be loaded in liquid form into the sample through-holes using capillary action, with typical volumes of the sample through-holes being in the range of from 0.1 picoliter to 1 microliter. The interior surfaces of the through-holes may also have a hydrophilic surface or be coated with a porous hydrophilic material, or as described above, be coated with a biocompatible material such as PEG, to enhance the drawing power of the sample through-holes, attract liquid sample and aid in loading. Kits for performing the assay may also be prepared, comprising one or more sample platen as described, the primers being affixed within the hydrophilic sample through-holes of the microfluidic array, and also comprising reagents required for the selective ligation and amplification assay. Target nucleic acid sequence(s) can then be added as desired to perform the assay. If not already provided with the kit, enzymes required to carry out the ligation and amplification reactions can also be added along with the target nucleic acid sequence(s).

EXAMPLES Example 1. Homogeneous detection of amplified target sequence Homogeneous detection of amplified target sequences may be carried out using a dye specific for binding to double-stranded DNA or RNA. Primers PI and P2, upstream and downstream primers, respectively, do not participate in amplification of target sequence, but rather, are responsible for identifying the target sequence containing a SNP. When either primer PI or P2 contains a match to the SNP of interest in the target sequence, ligation of PI and P2 occurs, and then primer P3, the general extension primer, amplifies the P1-P2 product. Consequently, concentrations of primers 1 and 2 are preferably optimized and adjusted to not interfere with exponential amplification of the target sequence such that only linear amplification of competing non-target sequences occurs. Examples of ds-DNA- and/or RNA-specific dyes that may be used include SYBR^® Green I and SYBR^® Green fl, YOYO^®-l, TOTO^®-l, POPO^®-3 (see Appendix A, attached hereto), ethidium bromide (EtBr) and any other dye providing adequate sensitivity and ease of detection of desired amplified product. In a particular embodiment, a sample target sequence of nucleic acid, optionally containing a single nucleotide polymorphism, is mixed with at least three primers - a first upstream primer having at least a portion of its 3 '-end substantially complementary to a first segment at a first end of the target sequence, a second downstream primer having at least a portion of its 5 '-end substantially complementary to a second segment at a second end of the target sequence, the 5'-end of the second primer being adjacent to the 3'-end of the first primer wherein a nucleotide complementary to the SNP of the target sequence is present at either the 3 '-end of the first primer or at the 5 '-end of the second primer, and a third general extension primer that is substantially complementary to a random sequence segment at the 3'-end of the second primer and to a substantially similar sequence at the 5'- end of the first primer. Additionally, at least four different nucleotide bases, a thermostable polymerase and a thermostable ligase are included in the reaction mixture, the thermostable polymerase preferably one that lacks 5' to 3' exonuclease activity, such as the Stoffel Fragment (see Appendix B, attached hereto). Examples of other thermostable polymerases which lack 5' to 3' exonuclease activity include Isis™ DNA polymerase, Pyra™ exo(-) DNA polymerase, and Q-BioTaq DNA polymerase (see Appendix C, attached hereto). Alternatively, the assay may use a thermostable polymerase that lacks 3' to 5' exonuclease activity, or a thermostable polymerase that lacks both 5' to 3' and 3' to 5' exonuclease activity. Examples of thermostable polymerases which lack 3' to 5' exonuclease activity include Taq polymerase, SurePrime™ Polymerase, and Q-BioTaq™ DNA polymerase (id.). An example of a thermostable polymerase which lacks both 5' to 3' and 3' to 5' exonuclease activity is Q-BioTaq DNA polymerase (id.). Addition of a dye specific for ds-DNA such as SYBR^® Green I, or specific for RNA such as SYBR^® Green π, allows detection of amplified product, by monitoring fluorescence emission of dye- bound nucleic acid product at ~520 nm(see Appendix D, attached hereto). As can be seen in Figure 1-A, a target nucleic acid may contain a SNP within the target sequence. Upon denaturation, Primer 1 (PI) and Primer 2 (P2) bind to the 3' to 5' strand of the target sequence, adjacent to the SNP. There may be a gap of several (approximately 2-4) bases between the 3 '-end of PI and the 5 '-end of P2, or there may be no gap. In Figure 1-B1, the base complementary to the SNP of the target sequence is at the 3 '-end of PI. Alternatively, the base complementary to the SNP of the target sequence may be at the 5'-end of P2, as shown in Fig. 1-B2. The third primer (P3) • contains a random sequence (RS) complementary to the random sequence of the 3 '-end of P2, such that after ligation of PI and P2, P3 binds and extends the ligated primer product, thereby amplifying the complementary strand (5'-3' strand) of the target sequence. As discussed above, a competing reaction may occur, such that primer P3 binds to primer P2 and extends this sequence to produce a linear product based on the P2 sequence. Preferably, concentrations of primers PI and P2 are adjusted to minimize the competing linear reaction. As shown in Figure 1-C, un-ligated primer PI extends the 3' - 5' strand of the target sequence. In another, preferred embodiment shown in Figure 2 (A - E), the first primer (PI) also has a random sequence at the 5'-end. When a primer containing the complement to the SNP, either PI on its 3'-end or P2 on its 5'-end (see Fig. 1-B), binds to the target strand (see Fig. 2-B), primers PI and P2 are ligated, and the third primer (P3) then binds to the 3'-end of the ligated P1-P2 product and produces the (3' to 5') P3-amplified strand (Fig. 2-C). At this point, primer P3 now also binds to the (3' to 5') P3-amplified product and produces the other (5' to 3') amplified product (see Fig. 2-D). Both target strands have now been produced, and can go on to yield exponentially amplified target sequence (Fig. 2-E1 and 2-E2). Additionally, detection with a fluorescent dye, such as SYBR^® Green I (SGI) may be done at temperatures above the T_m of the linear product, i.e., any product produced non-exponentially, thereby removing competing signal from any dye bound to linear product. SYBR^® Green I and other dyes that bind to double-stranded nucleic acids do not bind to nucleic acids above their T_ras because at those elevated temperatures, the nucleic acids are denatured. As seen in the cartoon of Figure 3, a dye such as SYBR^® Green I binds to double-stranded amplified target nucleic acid with a concomitant large increase in fluorescence. Although SGI is shown in Figure 3 as intercalating into the amplified target ds-nucleic acid, nothing in the figure is intended to suggest either an actual structure, or actual mode of binding, for SGI with ds-nucleic acids. Alternatively, the use of molecular beacon probes, having a fluorescent group on one end and a fluorescence-quenching group on the other, may be used. In this system, the molecular beacon remains quenched until being bound to amplified product (see, for example, Appendix E, attached hereto) because the molecular probe is typically in a hairpin conformation with the fluorescent group in close proximity to the fluorescence- quenching group, until the probe binds to the target amplified product (causing the hairpin structure to unfold, separating the fluorescent group from the quenching group). Examples of fluorescence-quenching groups appropriate for embodiments of the present invention include the dark quencher dabcyl, and the Eclipse™ Quencher from Epoch (id.). Examples of appropriate fluorescent groups that may be used in accordance with the present invention include Epoch's Yakima Yellow™ and Redmond Red™ (id.), and any other appropriate fluorescent dye whose fluorescence may be quenched to an appropriately positioned quencher molecule. In another embodiment, real-time amplification may be measured using a

TaqMan^® probe that is homologous to an internal sequence of the target nucleic acid, and having a fluorogenic 5'-end and a quencher 3'-end. During PCR amplification and extension, the quencher molecule is removed from the probe by 5 '-exonuclease activity, releasing the fluorescent reporter molecule from close proximity to the quencher molecule on the 3 '-end of the probe, thereby producing an increase in fluorescence emission as amplified product is produced (see Appendices F and G, attached hereto). In this system, a polymerase having 5' to 3' exonuclease activity is required. Another embodiment utilizes a detection method for real-time amplification measurement that involves the use of a pair of amplification primers, one of which matches the random sequence of primer 2. One of these primers in the pair is labeled with a fluorescent group that only fluoresces when incorporated into a PCR product, similar to Lux™ primers known in the art (see Appendix H, attached hereto). In such an embodiment, the fluorescent group is quenched by secondary structure before incorporation into double-stranded product, such that prior to incorporation, a sequence in the primer/probe binds to a complementary sequence in the primer/probe containing the fluorescent group, quenching the fluorescent group. In another embodiment, primers 1 and 2 are FRET partners, such that when hybridized to the amplified target sequence, produced only after primers 1 and 2 are ligated and amplified, they fluoresce (see

Appendices E and also A) and thus permit detection of amplified target sequence. In a preferred embodiment, fluorescence detection would be carried out above the either the Tm for primer PI, or above the T_m for primer P2, or alternatively be carried out above the T_ms of both primers PI and P2, to avoid background signal from possible hybridization of PI and/or P2 to amplified target. In another embodiment, primer may be designed to exponentially amplify target nucleic acid products that are distinguishable by an increase or decrease in melting temperature (T_m), wherein the exponentially amplified target sequence is either stabilized as indicated by an increase in T_m or de-stabilized, as indicated by a decrease in T_m, relative to the melting temperatures of linearly produced non-target product produced from non-ligated primers. Variability in the random sequence, or elsewhere in the primers, may be used to produce such exponentially amplified target nucleic acid sequence distinguishable by melting temperature from the linear product. In another embodiment, a probe specific for hybridizing across the ligation junction formed after ligation of the first and second primers may be used. Such a probe may have a hairpin conformation with a fluorescent reporter group on one end and a fluorescence-quenching group on the other end whereby no fluorescence occurs when the probe is not bound across the ligation junction. By optimizing reaction conditions, such as temperature and/or ionic strength, the hairpin would be stabilized by binding across the ligation junction, whereupon fluorescence would occur and emission could be monitored to detect amplified product.

Example 2. Single-tube reaction system for distinguishing SNPs One preferred embodiment of the present invention is the single-tube reaction system shown in Figure 4. Similar to the embodiments shown in Figures 1 and 2 and discussed above in Example 1, a three-primer system is utilized to identify a SNP of interest in a target sequence of nucleic acid. Again, there is an upstream primer and a downstream primer that bind to the target nucleic acid, flanking the SNP of interest. The 3'-end of the upstream primer may be directly adjacent to the 5'-end of the downstream primer, or there may be a gap of between about 1 to 4 bases between the 3 '-end of the upstream primer and the 5 '-end of the downstream primer. Either the 3 '-end of the upstream primer or the 5 '-end of the downstream primer may contain the complement to the SNP of interest in the target nucleic acid. Unlike the embodiments shown in Figures 1 and 2, however, the single-tube reaction system allows simultaneous single-tube identification and distinction between one or more SNPs of interest in one or more target nucleic acid sequences of interest. This is accomplished by using unique sequences in each of the random sequence regions of the upstream primer and the downstream primer (the two which ligate) and the general extension primer. As see in Figure 4A, a single-tube reaction system may contain a first upstream primer A-B with random sequence A, which identifies a first SNP of interest in a first target nucleic acid segment, and a second upstream primer F-E with random sequence F, which identifies a second SNP or interest in a second target nucleic acid segment, and a general extension primer with random sequence D' complementary to random sequence D present in downstream primer C-D, wherein C is common to both target nucleic acid segments. Upon successful identification and binding to a target nucleic acid having a SNP of interest, upstream primers A-B and/or F-E will be ligated to downstream primer C-D, creating ligation products A-B-C-D and/or F-E-C-D. If a gap is present between the 3'- end of the upstream primer and the 5 '-end of the downstream primer, the gap will first be filled in by a polymerase activity, followed by ligation to form the ligation products. Extension of both ligation products can then occur by general extension primer D', to produce extended products A'-B'-C'-D' and F'-E'-C'-D'. Next, hybridizable probe A with fluorophore 1 and hybridizable probe F with fluorophore 2, hybridize to extended products A'-B'-C'-D' and F'-E'-C'-D', respectively, which is followed by amplification such that each of the probes with its particular fluorescent tag is incorporated into amplified product (A-B-C-D or F-E-C-D), triggering fluorescence of either fluorophore 1 or fluorophore 2 or both. In this way, one or more SNPs may be identified and distinguished in a single-tube reaction system by monitoring the fluorescent signals of the two (or more) fluorophores upon incorporation into amplified product. In another embodiment, an alternative single-tube reaction system for identifying and distinguishing one or more SNPs in one or more target nucleic acid segments is shown in Figures 5A-5F. Figures 5A through 5C are identical to Figures 4A through 4C, in that upstream primers A-B and F-E, downstream primer C-D, and general extension primer D' are present in the single-tube reaction system. Again, either the 3'-end of the upstream primers may contain the complement to the SNP of interest in the target nucleic acids, or the 5'-end of the downstream primer may contain the complement to the SNP of interest in the target nucleic acids, and upon binding to the target nucleic acids, the two primers may be adjacent, or have a gap of about 1-4 bases between the 3 '-end of the upstream primer and the 5 '-end of the downstream primer, which must be filled by a polymerase, before ligation between the upstream and downstream primer can occur. As shown in Fig. 5D, however, the alternative single-tube reaction system does not use hybridizable probes A and F with fluorophores 1 and 2 to amplify target nucleic acid, but rather, uses regular primers A and F to amplify extended products A'-B'-C'-D' and F'-E'-C'-D' into amplified target nucleic acids products A-B-C-D or F-E-C-D. Such a system may be advantageous when a particular target nucleic acid does not amplify ' efficiently with hybridizable probes that have bulky fluorophores attached to them. In this alternative single-tube reaction system, the amplified target nucleic acids are detected after amplification, by additional fluorescent-tagged hybridizable probes hyb-A and hyb- F, which differ from regular primers A and F in that they are shorter, and have secondary structure that dissolve at lower temperatures than the annealing temperatures of primers A and F (or fluorescent probes A and F in Figure 4). This allows inefficient competition between hyb-A and hyb-F probes and regular primers A and F, in amplification of extended products A'-B'-C'-D' and F'-E'-C'-D' into target nucleic acid products A-B-C-D or F-E-C-D, but allows enough competing reaction to occur to measure fluorescence of fluorophores 1 and 2, thereby allowing detection and quantitation of amplified target nucleic acid product. Although use of a general extension primer such as D' that is complementary to a sequence D in segment C-D common to both target nucleic acid segments is convenient in the single-tube reaction systems described above and exemplified in Figs. 4 and 5, it is not required. It is envisioned that single-tube reaction systems could also be adapted for creating ligation products with with A-B and F-E using more than one extension primer simultaneously. The selectivity of the first primer A-B for the first SNP and the second primer E-F for the second SNP will ensure selective ligation, even with additional primers being used to generate the C-X product to be ligated. Upon successful identification and binding to a target nucleic acid having a SNP " of interest, upstream primers A-B and/or F-E will be ligated to downstream primer C-G and C-H, respectively, creating ligation products A-B -C-G and/or F-E-C-H. If a gap is present between the 3 '-end of the upstream primer and the 5 '-end of the downstream primer, the gap will first be filled in by a polymerase activity, followed by ligation to ■ form the ligation products. Extension of both ligation products can then occur by extension primers G' and H', to produce extended products A'-B'-C'-G' and F'-E'-C'-H'. As described above, one or more SNPs may be identified and distinguished in a single-tube reaction system by a) monitoring the fluorescent signals of two (or more) fluorophores upon incorporation into amplified product, or b) detecting fluorescent signals after amplification, by use of additional fluorescent-tagged hybridizable probes hyb-A and hyb-F.

Example 3. A nanoliter sampling array Another embodiment of the present invention encompasses a nanoliter sampling array. Any array presently available in the prior art may be used, but an array of particular utility, similar to that described in U.S. Provisional Application Serial No. 60/518,240, filed November 7, 2003, and US regular application serial no. 10/984,027 filed on November 8, 2004, both of which are hereby incorporated by reference herein, is one preferred array. In this particular embodiment, the array comprises a first platen having at least one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes. A target nucleic acid sequence is selected, and the array is prepared wherein each through-hole in the array contains at least a first primer having at least a portion of its 3'-end substantially complementary to a first segment at a first end of the nucleic acid target sequence and a second primer having at least a portion of its 5 '-end substantially complementary to a second segment at a second end of the nucleic acid target sequence, the 5 '-end of the second primer being adjacent to the 3 '-end of the first primer upon binding to the potential nucleic acid target sequence. Figure 4 shows such an array, known in the prior art. Array chip 10 typically may be from 0.1 mm to more than 10 mm thick; for example, from 0.3 to 1.52 mm thick, and commonly 0.5 mm. Typical volumes of the sample through-holes 12 could be from 0.1 picoliter to 1 microliter, with common volumes in the range of 0.2 to 100 nanoliters, for example, about 35 nanoliters. Capillary action or surface tension of the liquid samples may be used to load the sample through-holes 12. For typical chip dimensions, capillary forces are strong enough to hold liquids in place. Chips loaded with sample solutions can be waved in the air, and even centrifuged at moderate speeds, without displacing the samples. : To enhance the drawing power of the sample through-holes 12, the target area of the receptacle interior walls 42 may have a hydrophilic surface that attracts a liquid sample. Alternatively, the sample through-holes 12 may contain a porous hydrophilic materiel that attracts a liquid sample. In some embodiments, the sample through-holes in the array may be coated with a biocompatible material such as polyethylene glycol, and the primers may be affixed on, within or under the biocompatible material on the surface of the through-holes by drying the primers after application to the through-holes. To prevent cross-contamination (crosstalk), the exterior planar surfaces 14 of chip 10 and a layer of material 40 around the openings of sample through-holes 12 may be of a hydrophobic material. Thus, each sample through-hole 12 has an interior hydrophilic region bounded at either end by a hydrophobic region. The through-hole design of the sample through-holes 12 avoids problems of trapped air inherent in other microplate structures. This approach, together with hydrophobic and hydrophilic patterning enable self-metered loading of the sample through-holes 12. The self-loading functionality helps in the manufacture of arrays with pre-loaded reagents, and also in that the arrays will fill themselves when contacted with an aqueous sample material. Example 3. Method for identifying a SNP in a target sequence of nucleic acid. Yet another embodiment is a method for identifying a single nucleotide polymorphism (SNP) in a target sequence of nucleic acid. A target sequence of nucleic acid is identified, and primers are prepared according to standard methods, such that two primers, PI and P2, are designed to flank an internally-positioned SNP on one strand of the target nucleic acid sequence and are designed to be ligated with a thermally stable ligase. Primer PI and P2 are further designed such that the base complementary to the SNP in the target sequence is either on the 3 '-end of PI, or on the 5 '-end of P2. In this particular method, a nanoliter sampling array is used. The method comprises providing a first platen having a high-density microfluidic array of through-holes is provided wherein each through-hole of the array contains a first primer having at least a portion of its 3 '-end substantially complementary to a first segment at a first end of the target sequence, and a second primer having at least a portion of its 5 '-end substantially complementary to a second segment at a second end of the target sequence. Upon binding to the target sequence, the 5'-eήd of the second primer is adjacent to the 3'-end of the first primer. The method further comprises introducing a sample containing the target nucleic acid sequence with internal SNP into the array, and introducing reagents into the through- holes in the array wherein the reagents include a third primer having a random sequence capable of amplifying ligated primer P1-P2 product, a thermostable polymerase, a thermostable ligase, and at least four different nucleoside triphosphates. Additional steps in the method comprise thermocycling the array with primers, target nucleic acid, and reagents, and detecting the resulting amplified target nucleic acid sequence. Optionally, the thermostable polymerase may lack 5' to 3' exonuclease activity, or it may lack 3' to 5' exonuclease activity, or it may lack both 5' to 3' and 3' to 5' exonuclease activity. It is also envisioned that the detecting step may comprise the use of a dye specific for binding to double-stranded DNA or to RNA that fluoresces upon binding amplified target sequence. Suitable dyes include SYBR^® Green I, SYBR^® Green II, YOYO^®-l, TOTO^®-l, POPO^®-3, EtBr, and any other dye capable of providing low-sensitivity detection of amplified target sequence by fluorescence emission. Alternatively, detection may occur through the addition of probes specific for hybridization across the ligation junction of the ligated P1-P2 primer product, where such probes contain a fluorescent group and a fluorescence-modifying group such as a fluorescence quencher. In another alternative embodiment, detection may involve the use of a probe containing a fluorescent group and a fluorescence-modifying group such as a fluorescence quencher that is specific for hybridizing to a region of the target sequence. In this particular embodiment, the fluorescence-modifying group is excised upon extension of the probe, and the fluorescent group thus fluoresces, allowing detection of amplified product. Additional embodiments of the present invention include a kit for use in identification of amplified target nucleic acid sequences, wherein the kit provides a sample platen having one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes. In one particular kit each through-hole contains at least a first primer having at least a portion of its 3 '-end substantially complementary to a first segment at a first end of potential nucleic acid target sequence, a second primer having at least a portion of its 5'-end substantially complementary to a second segment at a second end of the potential nucleic acid target sequence, the 5 '-end of the second primer being adjacent to the 3 '-end of the first primer upon binding to the potential nucleic acid target sequence and a reagent platen having a high-density microfluidic array of through- holes with each through-hole containing a third primer that is substantially complementary to a random sequence segment at the 3 '-end of the second primer and to a substantially similar sequence at the 5 '-end of the first primer, at least four different nucleotide bases, a thermostable ligase and a fluorescent dye. In this particular embodiment, the reagent platen has a structural geometry that corresponds to the sample platen allowing delivery of reagent components and target nucleic acid sample to the primers in the sample platen. In some embodiments of the kit, the primers may be affixed on, within or under a biocompatible material such as a wax-like coating in the through- holes by drying the primers after being applied to the through-holes, wherein the biocompatible material may comprise, for example, a polyethylene glycol (PEG) material. To perform the selective ligation and amplification reaction for identification of an amplified target nucleic acid sequence, the user would merely add a sample containing the target nucleic acid, a thermostable polymerase, and optionally a buffer supplied with the kit to the through-holes.

Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage

Updated: August 30, 2003

Section 8.7 — Analysis of DNA Structure, DNA Binding and DNA Damage

Nucleic Acid Conformatioπal Analysis

A number of conventional dyes have been used to analyze nucleic acid conformation In vitro and In vivo'. • Acridine orange (A-1301, A-3568 ; Section 8.1) is one of the most popular and versatile fluorescent stains for histochemistry and cytochemistry and can provide a wide variety of information about the in situ content, molecular structure, conformation and environment of many nucleic acid-containing cell constituents-^^ « Fluorescence photobleaching of DNA that has been photolytically labeled with ethidium monoazide (E-1374, Section 8.1) permits measurement of slow reorientational motions.*® • The fluorescence intensity and binding affinity of the Hoechst dyes appear to be highly dependent on the sequence and conformation of the DNA base pairs. <f^*ϋ§ For example, staining by Hoechst 33258 (H-139S, H-3569; FluoroPure Grade. H-21491; Section 8.1) can discriminate parallel and antiparallel stem regions in hairpin DNA conformations, d& • The fluorescence lifetime of the PicoGreen dye (P-7581, P-1 495; Section 8.3) bound to single-stranded DNA is reported to be different when bound to double-stranded DNA.*¹©

We also anticipate that several of our cyanine dyes (Section 8.1) — in particular the SYTO dyes (Table 8.3) — may be useful in these applications because many of these stains appear to yield environment-sensitive rnetachromatlc shifts upon binding to nucleic acids. Fluorescence of the TOTO-1, YOYO-1, BOBO-1 and POPO-1 dyes (Table 8.2, Pimeric Cyanine Nucleic Acid Stains^') is dependent on nucleic acid secondary structure; a shift to longer-wavelength emission and a concomitant drop in quantum yield are observed upon binding of these dyes to single-stranded nucleic acids at high dye: base ratlos.Λ& Most of our unsymmetrical cyanine dyes show this spectral shift, and some show sequence selectivity in their fluorescence Intensity as well.

Examining the Behavior of Single Nucleic Acid Molecules

Once bound to nucleic acids, several of the cyanine dyes in Section 8.1 are so bright that they can be used to directly visualize single nucleic acid molecules in the fluorescence microscope (131, "El). The YOYO-1 and POPO-3 dyes (Y-3601, P-3584) dyes have also been used to follow the making and breaking of single chemical bonds. <IS A number of laboratories have taken advantage of the high sensitivity of these dyes to detect single nucleic acid molecules and to study biopolymer behavior: • Video microscopy has been used to observe relaxation of YOYO-1 dye-stained phage lambda DNA multlmers, after stretching in a fluid flovt,0t on a surface <f& or with optical tweezers. <^"$& TOTO-1 dye (T-3600) has also been used in this application._*^ • Individual YOYO-1 dye-ssDNA molecular complexes have been imaged in solution by Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage fluorescence video microscopy. <S3- • Molecular combing, a technique that uses a receding fluid interface to elongate DNA molecules for optical mapping of genetic loci, was developed using the YOYO-1 dye.*©F • Adsorption and desorptlon of single molecules of YOYO-1 dye-stained phage lambda DNA have been observed on fused-sllica and C_1B chromatographic surfaces.4S£ • The activity of a single ecBCD enzyme, which unwinds and separates the strands of dsDNA, has been studied using YOYO-1 dye-stained dsDNA in conjunction with optical tweezers and eplfluorescence microscopy.*^ • Our YOYO-1 dye (Y-3601) has been used to stain DNA manipulated in solution by changing electronic fields, a technique that could prove valuable In miniaturizing and automating analysis of DNA fragments. ® • Staining with the YOYO-1 dye (Y-3601) was used to observe the interaction of DNA with various liposomes rfϋr and to size plasmlds in a flowing stream.*^*® • The YOYO-1 dye was also used to detect radiation-induced double-strand breaks in individual electrostretched bacterial DNA molecules. S^ • Single-molecule imaging of nucleic acids stained with either YOYO-1 or POPO-3 or a combination of the two dyes through collection of the entire fluorescence spectrum of their complex has been reported. *SSfr • Highly sensitive sheath-flow techniques have also been developed for detecting and discriminating the size of single TOTO-1 dye-DNA molecular complexes.*® • Large fragments of DNA stained with our TOTO-1 dye (T-36O0) have been sorted by flow cytometry. This extremely rapid analytical method yields a linear relationship between the fluorescence intensity and the fragment size over a 10-50 kilobase pair range.d_f^°r • The POPO-1 (P-3580, Section 8.1) and POPO-3 (P-3584) stains have been used to sensitively detect single DNA fragments by flow cytometry using two-photon fluorescence excitation. ■*^*©^• • The POPO-3 dye (P-3584^*) has been used to study a single chemical reaction w!th,an individual DNA molecule. POPO-3 dye-stained DNA molecules stretched taught on a glass surface relax when a focused laser beam causes fluorescence-related breakage of the DNA backbone, forming a gap that is visible by fluorescence mιcroscopy.*ϊϋ^t • The TOTO-1 (T-3600), YOYO-1 fY-360 ^*), POPO-3 fP-3584) and SYBR Green I (S-7563, S^ 7567, S-7585) dyes have been used to visualize lambda DNA that has been stretched between beads with optical tweezers.*©',-*® • Fragment sizing on single molecules of dsDNA stained with our PicoGreen reagent has also been reported. <&*^■ • The SYTOX Orange dye (S- 1368) is the preferred dye for single-molecule sizing of DNA fragments by flow cytometry in an Instrument equipped with a Nd:YAG laser.idδfr • DAPI (P-1306, P-3571; FluoroPure Grade, D-21490) has also been employed to detect a single DNA molecule in solution <ά&t and by fluorescence microscopy <$_?^■ and to detect femtogra s of DNA in single cells and chloroplasts.*lEEr

The high affinity and bright fluorescence of other cyanine dlmers has allowed researchers to follow stained and transfected plasmlds or stained virus particles within a cell.<^"Sfr

DNA Binding Assays

Electrophoretic Mobility-Shift (Bandshlft) Assays

Bandshlft assays to analyze DNA-protein interactions are conventionally performed using radloactlvely labeled DNA fragments. However, use of our high-sensitivity fluorescent dyes makes these assays much simpler to perform and eliminates radioactive waste issues. For example, SYBR Green I nucleic acid gel stain (S-7567, S-7563, S-7585: SYBR Green I Nucleic Acid Gel Stain) has been used to post-stain gels after electrophoresls and can detect bound and unbound DNA fragments with high sensitivity <Kr (Figure 8.134). The SYBR Gold nucleic acid gel stain (S- 11494, "Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage

SYBR Gold Nucleic Acid Gel Stain) is potentially even more useful in bandshift experiments because of its higher sensitivity. Molecular Probes has made bandshlft assays easy and more convenient with our Electrophoretic Mobility-Shift Assay (EMSA) Kit (E-33075). Our EMSA Kit provides a fast and quantitative fluorescence-based method to detect both nucleic acid and protein in the same gel (fiS), doubling the information that can be obtained from bandshift assays. This kit uses two fluorescent dyes for detection — SYBR Green EMSA.nu leic acid gel stain for RNA or DNA and SYPRO Ruby EMSA protein gel stain for proteins. Because the nucleic acids and proteins are stained In the gel after electrophoresis, there is no need to prelabel the the DNA or RNA with a radioisotope, biotln or a fluorescent dye before the binding reaction, and therefore there is no possibility, that the label will Interfere with protein binding. Staining takes only about 20 minutes for the nucleic acid stain, and about 4 hours for the subsequent protein stain, yielding results much faster than radioisotope labeling (which may require multiple exposure times) or chemilumlnescence-based detection (which requires blotting and multiple incubation steps). This kit also makes it possible to perform ratiometric measurements of nucleic acid and protein in the same band, providing more detailed Information on the binding interaction. The signal from the two stains is linear over a broad range, allowing accurate determination of the amount of nucleic acid and protein, even in a single band, with detection limits of ~1 ng for nucleic acids and ~20 ng for protein . Both stains can be detected using a standard 300 nm UV illuminator, a 254-nm epi-illuminator or a laser-based scanner (.£1). Digital images can easily be overlaid for a two-color representation of nucleic acid and protein in the gel. The EMSA Kit contains sufficient reagents for 10 nondenaturing polyacrylamide minigel assays, including: • SYBR Green EMSA nucleic acid gel stain • SYPRO Ruby EMSA protein gel stain • Trlchloroacetic acid, for preparing the working solution of SYPRO Ruby EMSA protein gel stain • Concentrated EMSA gel-loading solution • lac repressor, a DNA-binding protein to be used as a control • lac operator, control DNA • Concentrated buffer for the lac repressoπoperator controls • A detailed protocol (Electrophoretic Mobility Shift Assay (EMSA) Kit)

Fluorescent dyes have also been used to stain the DNA fragments or proteins before electrophoresis. For instance, proteins or DNA labeled covalently with a reactive fluorescent dye (Chapter 1, Section 8.2) can be easily tracked during capillary electrophoresis to monitor DNA- protein Interactions.*^ High-affinity nucleic acid stains have also been used prior to electrophoresis, although they can potentially Interfere with protein binding and alter mobility on the gel. The ethidium homodimer-1 (EthD-1, E-1169? Section 8.1), YOYO-1 and TOTO-1 dyes have been shown by several laboratories to be useful tools for labeling DNA prior to electrophoresis in bandshlft assays. EthD-1 and TOTO-1 were used to examine interactions between the binding domain of the Kluyveromyces lactis heat shock transcription factor and its specific binding site. S YOYO-l dye has been used to study the association of £ coif RNA polymerase with DNA templates dϋr and the binding of a heat-shock transcription factor to its promoter.*-® All ten of our spectrally distinct (Figure 8.1), high-affinity dimeric cyanine dyes (Table 8.2) and the ethidium hσmodimers are potentially useful for multlcomponent analysis in this application.

DNA Binding Assays In Solution

Molecular beacons exploit fluorescence resonance energy transfer (FRET) to simplify detection of nucleic acid hybridization in solution (Section 8.5, Figure 8.104). This method has also proven useful for studying DNA-protein interactions in solution. Binding of a molecular beacon to lactic dehydrogenase separated the fluorophore from the quencher on the two ends of the labeled oligonucleotide, resulting in an increase in fluorescence. *fϊt The assay is sufficiently accurate to "Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage measure binding constants. A molecular beacon was also used to develop a solution-based binding assay for o_-CP₂, which is part of an RNA-binding complex.*®*-

Selective Cleavage of Nucleic Acids with a Chemical Nuclease

The thlol-reactive iodoacetamide of 1,10-phenanthrollne (P-6879, Section 2.3) is a useful adjunct reagent for bandshift assays. Conjugation to thiol-containlng llgands confers the rnetaJ-binding properties of this important complexing agent on the Itgand. For example, the covalent copper- phenanthrollne complex of ollgonucleotides or nucleic acid-binding molecules in combination with hydrogen peroxide acts as a chemical nuclease to selectively cleave DNA or R A.*^*® This reagent can also be conjugated to proteins to detect nucleic acid binding and targeted cleavage.*^**©

Assessing DNA Damage

Comet (Single-Cell Gel Electrophoresis) Assay to Detect Damaged DNA

The comet assay — or single-cell gel electrophoresis assay — is used for rapid detection and quantitation of DNA damage from single cells.*© The comet assay is based on the alkaline lysis of labile DNA at sites of damage. Cells are Immobilized in a thin agarose matrix on slides and gently lysed. When subjected to electrophoresis, the unwound, relaxed DNA migrates out of the cells. After staining with a nucleic acid stain, cells that have accumulated DNA damage exhibit brightly fluorescent comets, with tails of DNA fragmentation or unwinding ( ). In contrast, cells with normal, undamaged DNA appear as round dots, because their intact DNA does not migrate out of the cell. The ease and sensitivity of the comet assay has provided a fast and convenient way to measure damage to human sperm DNA,<S monitor the sensitivity of tumor cells to radiation damage *^"S8- and to assess the sensitivity of molluscan cells to toxins in the environment.^S^ The comet assay can also be used in combination with FISH to identify specific sequences with damaged D A.*®

Comet assays have traditionally been performed using ethidium bromide (E-1305, E-3565) to stain the DNA;*tS^" however, our YOYO-1 dye (Y-3601) increases the sensitivity of the assay eightfold compared to ethidium bromide and the fluorescence background from unbound YOYO-1 dye is negligible.*© Use of the SYBR Gold and SYBR Green I stains (Section 8.4) further improves the sensitivity of this assay.*®-

TUNEL Assay for In Situ Detection of Fragmented DNA

To detect fragmented DNA in labeled cells, terminal deoxynucleotidyl transferase (TdT) along with a fluorophore-, biotin-, or hapten-labeled dUTP can be added to cells. TdT adds the labeled nucleotide to all available 3'-ends — the more fragmented the DNA, the more 3'-ends are available and the brighter the fluorescent signal. Direct TUNEL assays using ChromaTlde BODIPY FL-14-dUTP (C-7614) to visualize DNA fragment ends are four times more sensitive than TUNEL assays using fluoresceln-labeled dUTP S* (fil). Terminal deoxynucleotidyl transferase (TdT)-catalyzed Incorp^'oratlon of bromo dUTP into nucleic acids of apoptotlc cells and detection of the incorporated BrdU with an antibody conjugate Is the basis of the APO-BrdU TUNEL Assay Kit fA-23210_f Section 15.5). Indirect TUNEL assays using probes such as biotlnylated dUTP or our ChromaTlde DNP-11- dUTP (C-7610, Section 8.2) allow for amplification of the signal with our fluorophore- or enzyme- conjugated streptavidin conjugates (Section 7.6. Table 7.20) or with antl-DNP antibody (Section 7.4). Several additional assays for apoptosls can be found in Section 15.5.

Microplate-Based Assays for DNA Damage

Abasic sites in DNA, generated spontaneously or caused by free radicals, ionizing radiation or mutagens like MMS (methyl methanesulfonate), are one of the most common lesions in DNA and Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage are thougnt to be important intermediates in mutagenesis. A quick and sensitive mlcroplate assay for abasic sites can be performed using ARP (A-10550, Figure 8.137), a biotlnylated hydroxylamine that reacts with the exposed aldehyde group at abasic sites. Biotlns bound to the abasic sites can be quantltated with our fluorescent- or enzyme-conjugated streptavldin complexes dSfr (Section 7.6, Table 7.20). ARP is permeant to cell membranes, permitting detection of abasic sites In living cells. *^*!_^■>

The PicoGreen reagent has also been used to simplify denaturation assays for DNA damage. Strand breaks in dsDNA that result from DNA damage can be quantified by measuring the relative amounts of ssDNA and dsDNA in a damaged sample. The relative amounts of dsDNA to ssDNA can be assessed by measuring the increase In abεorbance at 260 n or by separating the two forms, of DNA by alkaline sucrose gradient centrifugation,*6fr filters,*®* or hydroxyapatlte chromatography. <^*E* However, the absorbance-based technique suffers from low sensitivity and thus requires relatively large sample sizes Sfr and separation of ssDNA from dsDNA is laborious. This assay becomes much simpler and more sensitive using the PicoGreen dsDNA quantitatlon reagent (P- 7581, P-7589, P-11495, P-11496, R-21495; Section 8.3), which preferentially detects dsDNA In the presence of ssDNA.*!® The dye can be added directly to the sample and the fluorescence signal read on a fluorescence-based microplate reader. This method makes it possible to screen large numbers of very small samples in a high-throughput setting. The PicoGreen reagent was also used to develop a homogeneous PCR-based genotyping assay.*!© Because the products do not need to be run on a gel, the assay can be easily adapted for high throughput particularly using the RediPlate 96 version of the PicoGreen dsDNA quantitation assay (R-21495, Section 8.3).

Assays for Enzymes that Modify Nucleic Acids

Gel-Based Assays for DNase Detection

Our SYBR Green I stain (S-7563, S-7567, S-7585; SYBR Green I Nucleic Acid Gel Stain) has been used to develop DNase assays that show up to a 64-fold increase in sensitivity over similar ethidium bromide-based assays and up to 10,000-foId higher sensitivity than the traditional UV hyperchromicity assay. In a fast and simple assay, a single-length fragment of DNA can be incubated with the sample, followed by a short gel electrophoresis. Staining the gel with the SYBR Green I dye permits easy detection of less than 10^-5 Kunltz units (~5 pg) of DNase activity.*^ Even greater sensitivity can be achieved using the single radial enzyme diffusion (SRED) method, *ΪS!r In which the SYBR Green I stain is mixed with DNA in melted agarose and the mixture is poured Into a 2 mm thick slab. The sample to be tested Is poured into 1.5 mm circular wells punched out of the solidified agarose slab. As the sample diffuses through the agarose, the DNase degrades the DNA, creating dark circles around the sample well that do not show staining with the SYBR Green I dye when illuminated with UV light. The radius of these dark circles is proportional to the level of DNase activity. This method allows detection of as little as 2 x 10'⁷ units (~0.1 pg) of DNase I or 2 x 10^-6 (~0.9 pg) of DNase II. A third DNase assay — called the dried agarose film overlay (DAFO) method — uses the SYBR Green I stain to detect the presence of DNase activity in a polyacrylamide gel, allowing the Identification of heterogeneities in DNase species,*S This method allows the detection of 4 x 10^~6 units (~2 pg) DNase I or DNase II.

Solution-Based Assays for Nuclease Detection

Contaminating DNases are often responsible for poor resolution of DNA fragments, degradation of samples and nicking of supercoiled plasmids. Conventional DNase assays detect DNase activity by monitoring the increase in UV absorbahce that occurs when the base pairs unstack as the DNA is degraded. This absorbance method, however, is intrinsically insensitive as it requires large sample volumes and relies on small changes in absorbance. In contrast, our dyes for nucleic add detection show a tremendous fluorescence increase upon binding to nucleic acids, but their fluorescence is not affected by the presence of a large excess of a nucleotide or very short ollgonucleotides. Thus, nuclease activity can be easily and accurately measured by the decrease in fluorescence in the Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage presence of one of these dyes. For instance, the YOYO-1 nucleic acid stain (Y-3601) has been used in a fluorescence-based microplate assay for nuclease activity.*^- This assay takes advantage of the large fluorescence enhancement of the YOYO-1 dye upon binding to nucleic acids and corresponding lack of fluorescence in the presence of released nucleotides and very small nucleic acid fragments. Other dyes — in particular our PicoGreen dsDNA quantitation reagent (P-7581, E_: 11495; Section 8.3) — are likely to be more suitable for this assay. Similarly, use of the RiboGreen RNA quantitation reagent (R- 11490, R-11491; Section 8.3) should allow ultrasensitive detection of ribonuclease (RNase) activity.

Using a design similar to that of molecular beacons (Section 8.5), the stem sequence in an oligonucleotide hairpin loop can be modified to be a substrate for specific DNA cleavage agents, including nucleases. Dubbed a "break light," this substrate shows increased fluorescence as the cleavage agent breaks the DNA strand, separating the fluorophore from the quencher. sfr

An Assay for Reverse Transcrlptase Activity

The EnzChek Reverse Transcrlptase Assay Kit (E-22064) is a convenient, efficient and inexpensive assay for measuring reverse transcriptase activity (Figure 8.138). The key to this method is our PicoGreen dsDNA quantitation reagent, which preferentially detects dsDNA or RNA-DNA heteroduplexes over single-stranded nucleic acids or free nucleotides. In the assay, the sample to be measured is added to a mixture of a long poly(A) template, an oligo(dT) primer and dTTP. Reverse transcriptase activity in the sample results In the formation of long RNA-DNA heteroduplexes, which are detected by the PicoGreen reagent at the end of the assay. In less than an hour, samples can be read in a fluorometer or microplate reader with filter sets appropriate for fluoresceln (FITC). The assay is sensitive, detecting as little as 0.02 units of HIV reverse transcriptase, and has about a 50-fold linear range (Figure 8.139). Because it is much more rapid and less expensive than standard isόtopic assay or immunoassays, it is suitable for testing large numbers of biological samples. The assay's simplicity also makes It useful for automated high- throughput screening of reverse transcriptase inhibitors.

The EnzChek Reverse Transcriptase Assay Kit (E-22064) contains: • The PicoGreen dsDNA quantitation reagent • A lambda DNA standard • A poly(A) ribonucleotide template • An oligo(dT)16 primer • TE buffer, polymerization buffer and an EDTA solution • A detailed protocol (EnzChek Reverse Transcriptase Assay Kit)

Sufficient amounts of reagents are provided for approximately 1000 fluorescence microplate assays.

Telomerase

In a gel-based assay for detection of telomerase activity (the telomerlc repeat amplification protocol or TRAP) in human cells.and tumors, SYBR Green I dye staining was found to be more sensitive than silver staining and gave results comparable to those achieved with a radiolsotope- based TRAP assay.Λ& Moreover, unlike the silver stains, the SYBR Green I stain did not label proteins carried over from the reaction mixture. The SYBR Gold stain was also shown to be more sensitive than sliver staining In the TRAP assay, and much easier to use.<fδ- The SYBR Green I stain (S-7567, S-7563, s-7585) has also been used to develop high sensitivity assays to detect topoisomerase activity. *1# Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage

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fie Research ~ Abstracts: Lawyer et al.2 (4): 275

enzymat c character zat on o u - engt ermus aquaticus DNA polymerase and a truncated form deficient in 5* to 3¹ exonuclease activity

FC Lawyer, S Stoffel, RK Saiki, SY Chang, PA Landre, RD Abramson and DH Gelfand

Program in Core^'Research, Roche Molecular Systems, Alameda, California 94501.

The Thermus aquaticus DNA polymerase I (Taq Pol I) gene was cloned into a plasmid expression vector that utilizes the strong bacteriophage lambda PL promoter. A truncated form of Taq Pol I was also constructed. The two constructs made it possible to compare the full-length 832- arnino-acid Taq Pol I and a deletion derivative encoding a 544-amino- acid translation product, the Stoffel fragment. Upon heat induction, the 832-amino-acid construct produced 1 -2% of total protein as Taq Pol I. The induced 544-amino-acid construct produced 3% of total protein as Stoffel fragment. Enzyme purification included cell lysis, heat treatment followed by Polyrnh) P precipitation of nucleic acids, phenyl sepharose column chromatography, and heparin-Sepharose column chromatography. For full-length 94- kD Taq Pol I, yield was 3.26 x 10(7) units of activity from 165 grams wet weight cell paste. For the 61- fcD Taq Pol I Stoffel fragment, the yield was 1.03 x 10(6) units of activity from 15.6 grams wet weight pell paste. The two enzymes have maximal activity at 75 degrees C to 80 degrees C, 2-4 mM MgCE, and 10- 55 mM KG. The nature of the substrate determines the precise conditions for maximal enzyme activity. For both proteins, MgC12 is the preferred cofactor compared to MnC12, CoC12, and NiC12. The ^■full-length Taq Pol I has an activity half-life of 9 min at 97.5 degrees C. The Stoffel fragment has a half- life of 21 min at 97.5 degrees C. Taq Pol I contains a polymerization-dependent 5' to 3' exonuclease activity whereas the Stoffel fragment, deleted for the 5' to 3' exonuclease domain, does not possess that activity. A comparison is made among thermostable DNA polymerases that have been characterized; specific activities of 292,000 units/mg for Taq Pol I and 369,000 units/mg for the Stoffel fragment are the highest reported. I Genome Research -- Abstracts: Lawyer et al. 2 (4): 275 i wawfi

Return to F^*roduct Groups Polymerases for PCR The need for innovative application-specific thermostable DNA polymerases is

C CD More information growing rapidly. Qbiogene has developed a comprehensive selection of enzymes C/> covering all major PCR applications. Several of these polymerases are completely unique and offer important advantages. The tables below describe the major features and benefits of each enzyme. mΛ Taq DNA Polymerase For standard PCR applications - highly reliable, m well-established and widely used polymerase. m ^•»Q-BioTaq DNA Polymerase Recombinant truncated form of Taq DNA Polymerase lacking 5'-3'exo activity. ci Chemically modified Taq DNA Polymerase for m ^•"^■SurePrime Polymerase convenient "hot start" PCR. r σ> Hsis DNA Polymerase™ Highly accurate, high thermostabllity proofreading recombinant DNA Polymerase originally isolated from Pyrococcus abyδsii. Error rate is approximately 1 per 1.6 million base pairs per PCR cycle. ►Arrow Taq™ DNA Polymerase High performance mix for high sensitivity and long template amplification. Accuracy sufficient for most cloning needs. ►Pyra™ exo DNA Originally isolated from Pyrococcus abyssii but Polymerase lacking 5'-3' exo activity. Extremely thermostable. Recommended for standard PCR applications when very high resistance to

tnermocycimg is requireα. Taq DNA SurePrfme™ ∑sis DNA polymerase polymerase Arrow™ Q-bioTaq^*" Featαre/ General PCR Hot Start tipIex'PCR characteristic High Fidelity High Sensitivity/ Mul Long fragment PCR and RAPDs 5' -> 3' exonuclease 3' -> 5' exonuclease + + Error rate (10⁶) 24 24 0.6 S 24 Thermostabllity (half life at 95°C) 40 min 40 min 18h 40 min 80 min Residual polymerase activity at 25°C yes no yes yes yes Longest amplicons Up to 7 kb Up to 7 kb Up to 10 kb Up to 21 kb Up to 7 kb

C 3* - end dA dA blunt dA blunt mix dA CD Quality Control To ensure the highest standard of quality, each lot of thermostable DNA m polymerase is checked for activity, function and purity. Each thermostableΛ polymerase Is shipped with a lot-specific quality control datasheet. m m Absence of nic ases Confirmed by incubating increasing amounts of enzyme with supercoiled plasmid ci DNA (pBR322). The maximum number of units that results In no relaxation of the m supercoiled DNA, as visualized on an agarose gel, is stated on the lot-specific data r sheet. σ> Absence of endonuclease contamination Verified by an assay with Hind III/EcoR I fragments of DNA. Increasing amounts of enzyme are incubated with constant amount of substrate under appropriate incubation conditions. The maximum number of units that results in no degradation of the fragment pattern on agarose gel electrophoresis is stated on the lot-specific data sheet. Absence of 3' exonuclease activity- Confirmed on 32P Klenow labeled Hind III fragments of DNA. After incubation under appropriate conditions, the absence of released radioactive phosphate is checked by means of DE-81 adsorption. Absence of 5' exonuclease and 5' phosphatase activity

Demonstrated on 32P polynucleotide kinased Hind III fragments of DNA. After incubation under appropriate conditions, the absence of released radioactive phosphate is checked by means of DE-81 adsorption. Absence of ribonucleases Checked on 32P labelled RNA. After incubation under appropriate conditions, the absence of released 32P is checked using DE-81 adsorption. PCR assay 1 Carried out using human genomic DNA as a template (β-globin gene) with decreasing amounts of template DNA and decreasing units of DNA polymerase. A specific PCR product of 420 bp must be obtained.

<2 PCR assay 2 oo Carried out using phage DNA as a template ( DNA) with decreasing amounts of ff} template DNA and decreasing units of DNA polymerase. A specific PCR product of 500 bp must be obtained. c m Purity

< > w Verified by SDS-PAGE. m m ci ► Patent Information for PCR Process m r σ> Patent Information for PCR Process

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For general laboratory use. FOR IN VITRO USE ONLY. A

SYBR Green I Nucleic Acid Gel Stain

Highly sensitive fluorescent stain for detecting DIMA in agarose and polyacrylamide gels

Cat. No. 1 988 123 2 x 500 μl vx tmmtmiM Cat. No. 1 988 131 500 μl Store at -15 to -25* C

1. Product overview Product storage/ SYBR Green I Is supplied In an anhydrous DMSO stability solution and is shipped at ambient temperature.

Caution Because this reagent binds to nucleic acids, it should The unopened vial Is stable at -1 S to -25° C through be treated as a potential mutagen and used with the expiration date printed on the label. appropriate care, the DMSO stock solution shoutd be iltotø Allquote the stock solution In 50 μl allquots, handled with particular caution as DMSO is known to brown tubes should be used. facilitate the entry of organic molecules into tissues, When handling the DM SO stock solution, double gloves, protective clothing and eyewear should be worn and safe laboratory practices should be followed.

Contents

Handling protect from light

recommendations before opening, each vial should be allowed to warm up to 15-25° C and briefly centrifυged in a microfuge Test principle The dye exhibits a preferential affinity for nucleic acids to deposit the DMSO solution at the bottom of the vial. and its fluorescent signal Is largely enhanced when bound to DNA .(more than one-order of magnitude store aqueous stain solutions In polypropylene larger than the fluorescent enhancement or bound rather than glass, as the stain may adsorb to glass ethidium bromide). surfaces, dye Is not stable In water alone.

Application SYBR Green I dye Is a highly sensitive fluorescent stain for detecting nucleic acids In agarose and polyacrylamide gels (1,2). 2. Product characteristics The exceptional sensitivity of SYBR Green I stain makes it useful for those applications where the amount of DNA is limiting, Including the detection Sensitivity The detection limit using SYBR Green I is as low as of low-cycle number and low-target number DNA 100 pg per band of ds DNA uslnα 312 nm trans- amplification products; the detection and restriction illumination with the Luml Image?' F Instrument analysis of low-copy number of DNA and RNA vectors; from Roche Diagnostics (Cat No.2015170). and the detection of products of nuclease protection This is approximately 25 -100 times more sensitive than and bandshlft assays. ethidium bromide staining. SYBR Green I stain^'s superior sensitivity allows The detection limit for ollgonucleotldes stained replacement of radioisotope. in some applications, with SYBR Green I is below 50 pmol with e.g. RT-PCR. 312 nm translllumlnatlon (Luml-lmager Fl) or BteJg: SYBR Green I stain can also be applied as 25 nmeplillumlnatlon detection format In the UghtCyclβr Instrument But the provided concentration in DMSO Is not standardized Toxlclty The Ames mammalian mlcrosome reverse mutation for the precise quantification and detection of nucleic assay shews significantly less mutagenlclty of SYBR acids with the LlghtCycler. Please refer to specialized Green I than ethidium bromide (3,4) kits and reagents for this instrument Spectral SYBR Green I Is maximally excited at 497 nm and has

Sample material SYBR Green I can detect: characteristics secondary broad excitation peaks at 284 nm and 382 nm. The emission of DNA stained with SYBR • double stranded and single stranded DNA Green I Is centered at 520 nm. • RNA (with lower sensitivity); for RNA staining we recommend to use SYBR Green II Detection The spectral characteristics of SYBR Green I makes • Ollgonucleotldes it compatible with a wide variety of gel Imaging Instruments: Λfejβ.The detection limit for ollgonucleotldes stained with SYBR Green I is 1-2 ng with 254 nm * UVtraπs-lllumlπators epl-illumlnator or 312 nm translllumlnatlon, * UVepi-lllumlnators * argon Ion lasers.

Staining time Within 30 minutes, gels are ready to Image or photograph without destalnlng. flete Double stranded DNA-bound SYBR Green I stain fluoresces green under UV translllumlnatlon, Gels

Number of stains 500 μl stock solution Is sufficient to prepare a total of that contain DNA with single stranded regions may 5 liters of working solution, which can be used to stain show fluorescence that Is more orange tt»n green. more than 100 agarose or potyacryl mlπlgels. Advantage 3.2 Preparation of working solution Caution Since SYBR Green I binds to nucleic acids, It should be treated as a potential mutagen and used with appropriate care. The DMSO stock solution should be handled with particular caution as DMSO is known to facilitate the entry of organic molecules Into tissues. When handling the DMSO stock solution double ' gloves, protective clothing and eyewear should be worn and safe laboratory practices should be followed.^' Preparation of Dilute SYBR Green I stock solution 1:10000 In TE, TBE

SYBR Green or TAE buffer. The diluted solution has to be stored In working solutions polypropylene containers or bottles. HsS Staining with SYBR Green I is very pH sensMve. For optimal sensitivity, verify that the pH of the staining

3. Procedures and required materials solution at the temperature used for staining Is ^•between 7.5 and 8 (preferably pH 8.0).

3.1 Before you begin Disposal As with all nucleic acid stains, solution of SYBR Green I

Prestained gels We do not recommend preparing prestained gels with should be poured through activated charcoal before SYBR Green I stain more than 1-2 days in advance. disposal. The charcoal must then be Incinerated to destroy the dye. One gram of activated charcoal easily Gels previously stained with ethidium bromide can absorbs the ye from 10 liters of freshly prepared subsequently be stained with SYBR Green I following working solution.- the standard protocol for poststainlng. There may be some decreases in sensitivity when compared to a gel stained only with SYBR Green I. 3.3 Staining DNA following electrophoresis

Electrophoresis Perform electrophoresis on an agarose gel or denaturing polyacrylamide gel using: Additional UV trans- or epi-lllumlπators, or respective Imaging TBE [89 M Trls base, 89 M boric acid, 1 mM EDTA, equipment and instruments, such as Luml-lmager F1, argon ion pH 8] or reagents required lasers or respective Imaging instrument TAE [40 mM Trls-acetate, 1 M EDTA, pH 8] buffer. • clear polyproylene container for staining Note: Do not add any SDS to the electrophoresis • TBE buffer or buffer as this will dramatically reduce staining • TAE buffer efficiency. Procedure Please refer to the following table for the protocol,

Additional buffers TBE [89 mM Trls base, 89 M boric acid, required for 1 mM EDTA, pH B] or Note: For reaching sharp bands and low background staining stain the gel directly after electrophoresis. • TE [10 M Tris-HCI, 1 M EDTA, pH 8] or • TAE [40 mM Tris-acetate, 1 M EDTA, pH 8] buffer. Step Action Place the gel in a fitting polypropylene container.

Handling In the following table please find information about Note: There is no need to wash urea or instructions for working conditions for successful! staining. formaldehyde out of the gels prior staining. proper staining Add enough staining solution to cover the gel. Protect the staining container from light by covering it with aluminum foil or placing in the dark. Stain the gel for approx.30 min under constant agitation gently at 15-25' C. Illumination of the stained gel:

Photograph the gel with Polaroid 667 black and white print film using a SYBR Green gel stain photographic filter. Note; Stained gels have negligible background fluorescence, allowing long film exposure using an f-stop o .5 Is adequate.

Roche Molecular Biochexiήcals 3.4 Precastlng gels with SYBR Green I stain 3.5 Staining DMA before Electrophoresis

General In the following. table please find the features of General See references (5,6) for general methods on how to precast gels. stain DNA before electrophoresis. It may be necessary to optimize the protocols In these references for the specific application. We have stained 1 mg molecular weight-marker DNA with 1:10000 dilution of SYBR Green I in a total volume of 16 ml. SYBR Green I has also been tested as a prestainlng label for DNA templates In bandshift assays, and has been prove to be useful in this application. Procedure Please refer to the following table.

Procedure The final dilution of the SYBR Green I is best determined empirically, as there may be some non linear ef ects on the migration of different fragment

size. Step Action Dilute SYBR Green I stock reagent 1:10000 into the gel solution Just prior to pouring the gel. 3.6 Removing SYBR Green I stain from double-stranded DNA The liquid should be as cool as possible when the dye Is added. Procedure Note, Boiling and near boiling temperatures At least 99.9% of SYBR Green I can be removed from destroy the ability of SYBR Green I to stain double-stranded DNA by simple ethanol precipitation. nucleic acid. Do not heat SYBR Green 1 In the microwave. Follow gel electrophoresis as usual. Illumination of the stained gel:

Photograph the gel with Polaroid 657 black and white print film using a SYBR Green gel stain photographic filter. Note: Stained gels have negligible background fluorescence, allowing long film exposure using an f-stop of 4.5 Is adequate.

Roche Molecular Biochemicals 4* References E-mail Adress Country 1 Schn-ebergβr. C. et al.1895. PCR Methods & App. 4.234-238. argentlnalJloch8m@roche.com Argentina 2 Stager, V., et at 1994. Blomed Products 19, 68. b|ocherr_au^@rocfw.com Australia 3 MutattanRes. 113,173 (1983) GerhardMuehlbauer@roche.com Austria 4 PWS10, 2281 (1973) bloctiefnboSrocho.com Belgium 5 Mθtti Enz ttol 217, 14 (1993) btαchemcaOroche.corn Canada 6 Nudelc Adds Res.20, 2803 (1992) blocriem.cn@rocrie.corn China Blochemcynlcosla^@roche.com Cyprus Bmcomp@mbox.votcz Czech Republic

¹¹ SYBR Green I Is a trademark of Molecular Probes, Inc., Eugene, OR, USA. blochenUi@oriola.fl Finland ^*"^* Luml-lmager Is a trademark of a Member of the Roche Group. blochem.fr^@roche.coro France blochemlnfade@roche.com Germany »ubanegln@lstn.lros com Iran agetttek@tbm.net Israel itblochem^@roche.com Italy

4.1 Related products tønWΦIo@cet.coJp Japan Bmskorea@cholllaruιet Korea blochemInront@oxha.com Netherlands bIochemJiz@roche.com New Zealand medlnor@medlnor.no Norway blochenvpt@roche.com blochern.sg@roche.com sou-t--frlcaJ)Iobotπn@roche.com South Africa bIochem.es^@roche.com Spain Wochemse^@roche.com Sweden B!ocr«m!π|».CH@rocrιβ.com Switzerland ukblochem@roche.com United Kingdom blochenrts-is@rocha.com USA

Woohe ts-OwβrocheΛom All ether countries http://blochem.roche.eom/pack-insert/1988123a.pdf Argentina 541 954 5555; Australia (02) 9899 7999; Austria (01) 27787; Belgium (02) 247 4930; Brazil +55 (11) 36663565; Canada (450) 6867050; (800) 361 2070; Chile 0056 (2) 22 33737 (central) 0056 (2) 2232099 (Exec); China 8621 64275586; Columbia 0057-1 -3412797; Czech Republic (0324) 46 54. 5β 71-2; Denmark +45 363 999 58; Egypt 202 348 1715; Finland (09) 429 2342; France 0476763087; Germany (0621) 759 8568; Greece 3 (01) 67 40238; Hang Kong (852) 24857596; India 0031 (22) 431 2312; Indonesia 62 (021) 2523820 ext 755; Iran 009821208 2266 + 0098218785656; Israel 972 36 49 31 11; Italy 039247 4109-4131: Japan 03 3432 3155; Kenya 00254-2-74 4677: Korea 82-2-3471-65Q0; Kuwait 00965-4832600; Luxembourg 00352-4824821: Malaysia 60 (03) 7555039; Mexico (5) 227 8967; Netherlands (036) 5394911; New Zealand (09) 2764157; Nigeria 00234-1-960984; Norway 22076500; Philippines (632) 8107246; Poland +48 (22) 226684305; Portugal (01) 4171717; Republic ol Ireland 1 800409041; Russia (49) 6217598636 Fax: (49) 6217S98611; SeutOa Arabia +966 1 4010333; Singapore 0065 2729200; South Africa (011) 8862400; South Eastern Europe (01) 27787; South Korea 025696902; Spain (93) 201 4411; Sweden (08) 4048800; Switzerland +41 (41) 7996161: Taiwan (02) 736 125; Thailand 66 (2) 27407 08 (12 line); Turkey 00902122163280; United Kingdom (0800) 521579: USA (800) 4285433.

Roche Diagnostics GmbH Roche Molecular Blochemlcals

Sandhofer Strasse 116 ^• D-68305 Mannheim Germany tet. Research Glen Report βppwd'X £ Redmond Red™And Yakima Ye!low™Dyes, And Eclipse™ Non-Fluorescent Quencher

Glen Research is happy to confirm our agreement with Epoch Biosciences, Inc., announced on Februaiy 6, 2002, to distribute several of Epoch's proprietary products designed for the synthesis of novel DNA probes. Initially we will provide products based on Epoch's Redmond Red™ and Yakima Yellow™ fluorophores and Eclipse™ non-fluorescent quencher, which will be described in detail in this article. e will also supply PPG, a modified nucleoside, which is covered in the article on Page 8 of this newsletter. It is a pleasure for us to expand our relationship with Epoch by helping to provide broad access for these compounds to research markets worldwide. i As part of the preamble to this article, it is instructive to quote from the Epoch press release, which positions these products perfectly. They note that the products covered by this agreement are as follows: ¹ "all are innovative components for DNA probes, the workhorses of genetic analysis. Probes hybridize or bind to target DNA and then provide a si nal so

nτ«l*a_$ϋk (n.) 3_poch Fluorophores and Quencher The use of fluorescent tags as an alternative to radiolabels in DNA probes and primers has blossomed over the years. Fluorescence is safely measured with inexpensive instrumentation and it is very straightforward to multiplex assays for exceptionally high throughput. Molecular beacon and fluorescence resonance energy transfer (FRET) probes can be used in assays, which can be carried out in closedjybe formats with less sample handling at higher

throughput. These probes are also suitable for use in techniques that include amplification of the target DNA. Molecular beacon and FRET probes require efficient quenching until the probe is hybridized to the target. Molecular beacon probes are hairpin structures wherein the fluorescence is quenched by the proximity of the fluorophore to the quencher molecule. When the probe hybridizes to the target, it becomes linear, quenching is disrupted, and the probe fluoresces ready for detection. In FRET assays, when the probe is hybridized to the target, it is digested by nuclease activity in the polymerase being used for amplification of target copies. The fluorophore, released from the target and separated from the quencher, is now highly fluorescent and ready for detection^". Fluorophore/quencher pairs can be chosen based on spectral properties - the emission of the fluorophore should overlap the absorption of the quencher. The quencher may absorb its partner's fluorescence and emit the fluorescence at a new wavelength or, in the case of a non-fluorescent quencher, as heat. The new Epoch products offer two new fluorescent dyes, available immediately as phosphoramidites and supports, as shown in Figure 1 ; Their absorbance and emission characteristics are shown in Figure 2. Yakima Yellow has an absorption maximum at 530 nm and emission maximum at 549 nm, while Redmond Red's absorption and emission maxima are at 579 nm and 595 nm, respectively.

Measured in o.i M sodium phosphate. As indicated, Redmond Red is pH sensitive? Yakima Yellow is not and has the same extinction coefficient at pH 7 as pH 9. The QYs (quantum yield of fluorescence) were determined in 0.1 M Tris-HCI pH 7.4 relative to reference dyes using the dye-labeled T12 oligo. For Yakima Yellow, the reference dye was carboxy tetramethylrhoda ine in methanol. For Redmond Red, the reference dye was sulforhoda ine 101 in EtOH. Over the years, the dabcyl molecule has proved to be an excellent universal non-fluorescent (dark) quencher for molecular beacon probes. In addition, dabcyl is a very stable molecule and synthesis of doubly-labelled probes containing a dabcyl quencher is quite straightforward. The mechanism of quenching relies on the close proximity of the fluorophore to the dabcyl group, generally called static quenching, which is independent of spectral overlap between fluorophore and quencher. However in FRET probes, dabcyl's ability to act as a dark quencher is limited by its absorption spectrum to use with dyes emitting at 400 - 550 nm. Other fluorescent quenchers can be used to cover a broad spectrum of dyes, but synthesis of the doubly-labelled probes is made much more difficult by the lack of stability of some of the potential candidates to the conditions of oligo deprotection. These probes are best c

00 prepared in a two-step process requiring post-synthesis conjugation of the second dye, usually with purification problems. The Eclipse Quencher from Epoch solves most of the problems inherent in the synthesis of molecular beacon and FRET probes. The Eclipse molecule is highly stable and can be used safely in all common oligo deprotection schemes. The absorption maximum for Eclipse Quencher is at 522 n , compared to 479 nm for dabcyl. In addition, the structure of the Eclipse Quencher (Figure 1) is substantially more electron deficient than that of dabcyl and this lead m to better quenching over a wider range of dyes, especially those with emission maxima at longer wavelength (red shifted) such as Redmond Red and Cy5. A simple experiment, using a molecular beacon probe, showed that quenching of the fluorescence of Cy5 was 53% more efficient with Eclipse Quencher than with dabcyl. Epoch researchers conducted a comparisonl of the quenching ability of the Eclipse Quencher relative to dabcyl in an m m _: enzymatic digestion assay designed to simulate the efficiency of quenching of FRET probes. Over the range of fluorophores from FAM (520 nm) to Cy5 1(670 nm), Eclipse Quencher outperformed dabcyl in a measurement of signal to background ratio (signal, defined as fluorescence after digestion, divided

7i [by background, defined as initial fluorescence). The improvement was most marked for the higher wavelength dyes. In addition, with an absorption range c jfrom 390 nm to 625 nm (Figure 3), the Eclipse Quencher is capable of effective performance in a wide range of colored FRET probes. m jAs is normal these days, the use of these products is restricted and users should heed the following qualification statement: r σ> i'These Products are for research purposes only, and may not be used for commercial, clinical, diagnostic or any other use. The Products are subject to proprietary rights of Epoch Biosciences, Inc. and are made and sold under license from Epoch Biosciences, Inc. There is no implied license for jcommercial use with respect to the Products and a license must be obtained directly from Epoch Biosciences, Inc. with respect to any proposed ₌commerciaI use of the Products. "Commercial use" includes but is not limited to the sale, lease, license or other transfer of the Products or any material Tberived or produced from them, the sale, lease, license or other grant of rights to use the Products or any material derived or produced from them, or the ¹ use of the Products to perform services for a fee for third parties (including contract research)." ϊtedmond Red, Yakima Yellow, and Eclipse are trademarks of Epoch Biosciences, Inc. FIGURE 3: ABSORPTION SPECTRUM OF 3'-ECUPSE-LABELLED DT12 OLIGO

m Reference: m ^* (1) E.A. Lukhtanov, M. Metcalf, and M.W. Reed, American Biotechnology Laboratory, September, 2001. ci ORDERING INFORMATION m Please contact GlanRβsβareh if you have any questions or commentsl r 09/17/2001 00:00:01 I http://w w.olenres.com GlenReDorts/GR15-11.html σ> (Click link to bring page out of frameset for bookmarta^'ng.)

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TaqMan® Principles frpptrtiiX f-

Principle

Real-Time PCR Taqman

Append? X G~

Taqman Information

OVERVIEW Real-time quantitative PCR is a powerful tool that can be used for gene expression analysis, genotyping, pathogen detection/quantitation, mutation screening and DNA quantitation. At the BRC, we use the ABI Prism 7900 Real Time Quantitative PCR instrument (TaqMan®) to detect accumulation of PCR product, allowing easy and accurate quantitation in the exponential phase of PCR reactions. The ABI 7900 instrument continuously measures PCR product accumulation using a dual-labeled flourogenic oligonucleotide probe called a TaqMan® prbbe. This probe is labeled with two different flourescent dyes, the 5' terminus reporter dye and the 3' terminus quenching dye. The sequence of the oligonucleotide probe is homologous to an internal target sequence present in the PCR amplicon. When the probe is intact, energy transfer occurs between the two flourophors, and the fluorescent emission is quenched. During the extention phase of PCR, the probe is cleaved by 5' nuclease activity of Taq polymerase. Therefore, the reporter is no longer in proximity to the quencher, and the increase in emission intensity is measured. The ABI 7900 Prism software examines the fluorescence intensity of reporter and quencher dyes and calculates the increase in normalized reporter emission intensity over the course of the amplification. The results are then plotted versus time, represented by cycle number, to produce a continuous measure of PCR amplification. To provide precise quantification of initial target in each PCR reaction, the amplification plot is examined at a point during the early log phase of product accumulation above background (defined as the threshold cycle number or CT). Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each well. Primers, Probes, and Reagents It is essential to have a well thought out experimental design for Real Time PCR. Good primer and probe design is imperative. The BRC will design your probes and primers using Primer Express, the industry gold standard. Primers should be synthesized and purified at the BRC. This service is charged at our consultant rate of $50/hr. We require purified primers. Probes should be synthesized by Biosearch Technology. (www.biosearchtech.com). Black hole quench probes give the most consistent data. Average probe cost is $250. If you plan to perform your own Taqman® reactions, Applied Biosystems provides a number of kits specific to applications. See their web site www.AppliedBiosystems.com.

Facility Acknowledgement ReHj^'-tlnϊe^' PCR^'Taqman

The Taqman® facility requests an acknowledgement in the Methods section of any publications resulting from this data. An example is "Real time quantitative PCR was conducted by the Biomolecular Resource Center at the University of California, San Francisco." Additionally, if your project required special attention by a specific person at the BRC, an example would be "Technical expertise was provided by (specific name of BRC personnel) of the Biomolecular Resource Center at the University of California, San Francisco."

Biomolecular Resource Center Genetic Analysis Facility UCSF, Science 983, San Francisco, CA 94143-0541 Phone: (415) 514-0101 x1; FAX: 502-7649 Email: dna@cgi.ucsf.edu

ty tnJ/X H

Instruction Manual

LUX Fluorogenic Primers

For real-time PCR and RT-PCR

Version E

22 September 2003

25-0546 PAGE INTENTIONNALLY LEFT BLANK

Table of Contents

Introduction 1

Designing and Ordering Custom LUX™ Primers 3

Certified LUX™ Primer Sets for Housekeeping Genes 5

Storing and Reconstituting Primers 5

Real-Time PCR 7

Multiplex Real-Time PCR 11

Two-Step Real-Time RT-PCR 12

One-Step Real-Time RT-PCR 14

Troubleshooting 18

Accessory Products 19

Purchaser Notification '. 20

Technical Service •• 21

References

PAGE INTENTIONNALLY LEFT BLANK

Introduction

Overview LUX™ (Light Upon extension) Primers are an easy to use, highly sensitive, and efficient method for performing real-time quantitative PCR (qPCR) and RT-FCR (qRT-PCR). LUX™ Primers combine high specificity and multiplexing capability with simple design and streamlined protocols. LUX™ Primers require no special probes or quenchers, and are compatible with melting curve analysis of real-time qPCR products, allowing the dif erentiation of amplicons and primer di er artifacts by their melting temperatures. You can-custom- design LUX"' Primers™ from a target sequence using Web- or desktop-based software, or order predesigned and validated Certified LUX™ Primer Sets for Housekeeping Genes. Each primer pair in the LUX™ system includes a fluorogenic primer with a fluorophore attached to its 3' end, as well as a corresponding unlabeled primer. The fluorogenic primer has a short sequence tail of 4-6 nucleotides on the 5' end that is complementary to the 3' end of the primer. The resulting hairpin secondary structure provides optimal quenching of the fluorophore (see the figure below). When the primer is incorporated into the double- stranded PCR product, the fluorophore is dequenched and the signal increases by up to 10-fold.

LUX Primer Relative fluorescence; Reaction

Single-stranded primer

Extended primer (double-stranded DNA)

Labeling Each fluorogenic LUX™ primer is labeled with one of two reporter dyes — FAM (6-carboxy-fluorescein) orfOE (6-carboxy-4', 5'-dichIoro-2', 7'-dimethoxy- fluorescein). Additional reporter dyes will be available in the future.

Continued on next page Introduction, Continued

Applications LUX™ Primers can be used in real-time PCR and RT-PCR to quantify 100 or fewer copies of a target gene in as little as 1 pg of template DNA or RNA. They have a broad dynamic range of 7-8 orders. Multiplex applications use separate FAM and JOE-labeled primer sets to detect two different genes in the same sample. Typically, a custom-designed FAM-labeled primer set would be used to detect the gene of interest, and a JOE-labeled Certified LUX™ Primer Set would be used to detect a housekeeping gene as an internal control.

Instrument LUX™ Primers are compatible with a wide variety of real-time PCR Compatibility instruments, including but not limited to the ABI PRISM^® 7700/7000/7900 and GeneAmp^® 5700, the Bio-Rad iCycler™, the Stratagene Mx4000™, the Stratagene Mx3000™, the Cepheid Smart Cycler^®, the Corbett Research Rotor-Gene, and the Roche LightCycler^®. ABI PRBM is a registered trademark of Applera Corporation. GeneAmp is a registered trademark of Roche Molecular Systems, Inc. LightCycler is a registered trademark of Idaho Technologies, Inc. iCycler, Mx 000, Mx300O, Rotor-Gene, and Smart Cycler are trademarks of their respective companies.

Designing and Ordering Custom LUX™ Primers

LUX Designer To design and order custom LUX™ Primers for your genes of interest, visit the Primer Design I vitrogen LUX™ Web site at www.invitrogen.com/LUX and follow the link to Software the LUX™ Designer software. The software is available as either a Web-based application or a Microsoft* Windows^®-compatible download. Follow the step- by-step instructions in the software to submit your target sequence and generate primer designs. LUX™ Designer will automatically generate one or more primer designs based on each sequence you submit and the selected design parameters. The design software includes algorithms to minimize primer self-complementarity and interactions between primers. It also assigns rankings to the generated designs — based on primer melting temperature, hairpin structure, self- annealing properties, etc. — to aid in selection. When the designs have been generated, you can review them, select a design, select the fluorophore labels, and place your order.

Guidelines for When you submit a target sequence containing your gene of interest, keep in Submitting a mind the following design criteria: "Target Sequence • The optimal amplicon length for real-time PCR ranges from 80 to 200 bases. You can specify a nύnimum, optimal, and maximum amplicon length when you submit the sequence. • The target sequence should be at least 10 bases longer than the minimum amplicon size you select. The longer the sequence, the more likely that an optimal primer design can be developed. • The sequence must contain only standard lUPAC (International Union of Pure and Applied Chemistry) letter abbreviations. • When you select the design parameters, the default melting temperature range is 60-68°C. Do not change this default unless the design engine finds no primers in this range. For primers in this range, PCR annealing temperatures from 55° to 6i C are appropriate. When you first submit a sequence, the Disable Score-Based Rejection checkbox should not be checked; the resulting scores provide an important measure of primer suitability. Scores in the range of 0.0-4.0 are acceptable. If no primers with a score of 4.0 or lower can be generated from a sequence, you can disable score-based rejection and redesign the primers. Note that if you select a primer with a higher score, the efficiency of the reaction may be less than optimal. See the LUX™ Designer Help for additional guidance.

Selecting a Primer After you submit your sequence, LUX™ Designer will first generate one or Design more designs for the labeled primer. The labeled primer can be either the forward or the reverse primer. After you select a design for the labeled primer, you will be prompted to select a design for the corresponding unlabeled primer. Continued on next page Designing and Ordering Custom LUX ™ Primers, Continued

Selecting Labels After you have selected a primer set (labeled and unlabeled) for a particular sequence, you can specify the particular label and synthesis scale. Custom LUX™ Primers are provided in 50 nM or 200 nM synthesis scale. When selecting labels in a multiplex reaction, we recommend using the FAM label or your gene of interest and the JOE label for the housekeeping gene that you will use as the internal control. Certified LUX™ Primer Sets for Housekeeping Genes are recommended for Hie JOE-labeled control gene.

Placing^'the Order After you have selected the label and synthesis scale, you can submit your order to Invitrogen using the Web site or by e-mail or fax. Each.primer order will be shipped directly from Invitrogen's Custom Primer Facilities. Labeled primers are supplied in an amber tube; unlabeled primers are supplied in a clear tube. Each primer ordered from ^"tnvitrogen's Custom Primer Facilities comes with a Certificate of Analysis (COA) verifying the amount and sequence.

Product Custom LUX™ Primers are tested post-synthesis by optical density (OD) ratio Qualification measurements and mass spectroscopy to ensure efficient dye labeling and correct molecular weight and composition. See the Certificate of Analysis shipped with each primer for more information.

Storing and Reconstituting Primers

Primer Storage and Store primers at ~20°C in the dar LUX™ Primers are stable for: Stabⁱlity _# >12 months when stored at -20°C in lyophilized form. • >6 months when stored at -20°C in solution. Stability can be extended by storing at ~70°C.

Reconstituting Custom LUX™ Primers are provided lyophilized in 50-nmole or 200-nmole Primers synthesis scale. To reconstitute primers, centrifuge the tube for a few seconds to collect the oligonucleotide in the bottom of the tube. Carefully open, add an appropriate volume of TE buffer or ultrapure water, close the tube, rehydrate for 5 minutes, and vortex for 15 seconds. We recommend that you rehydrate primers at concentrations greater than 10 μM. To prepare a 100 μM primer stock solution, multiply the primer amount in nmoles by ten to determine the volume of diluent in ul. After reconstitution, store the primer stock at-20°C in the dark, where it will be stable for 6 months or more.

TM Certified LUX Primer Sets for Housekeeping Genes

Certified LUX™ Certified LUX™ Primer Sets for Housekeeping Genes are predesigned primer Primer Sets for sets for genes that are commonly used as internal controls for normalizing Housekeeping real-time RT-PCR experiments. These primer sets have been optimized and Genes functionally validated to provide accurate, reproducible results using standard LUX™ protocols. They are supplied ready to use in TE buffer. Each Certified LUX™ Primer Set includes a FAM- or JOE-labeled LUX^*" primer and a corresponding unlabeled primer. Each primer (labeled and unlabeled is supplied at 100 μl and a concentration of 10 μM. Available sets are listed below. For additional information, visit www.mvittogen.com/LUX.

Real-Time PCR

Introduction This section provides guidelines and protocols for performing real-time PCR using LUX™ Primers.

Template The target template for real-time PCR is linear single-stranded or double- Specifications stranded DNA, cDNA, or circular DNA (such as plasmids). The amount of DNA typically ranges from 10² to 10⁷ copies or 1 pg to 10 μg of template. Seepage 12 for instructions on generating cDNA using reverse transcription as part of two-step real-time RT-PCR.

Primer For optimal PCR conditions, primer titrations of 50-500 nM per primer are Concentration recommended. The sample reactions on pages 9-10 use 200 nM of each primer, equivalent to 1 μl of a 10 μM primer solution.

Magnesium The optimal M "^* concentration for a given target/primer /polymerase Concentration combination can vary between 1 mM and 10 mM, but is usually in the range of 3 mM. See the sample reactions on pages 9-10.

dNTP The optimal concentration of dATP, dCTP, dGTP, and dTTP is 200 μM each. If Concentration dUTP is used in place of dTTP, its optimal concentration is 400 μM.

Enzyme We recommend using a "hot-start" DNA polymerase, preferably one that has Specifications been optimized for real-time PCR. Platinum^® Quantitative PCR SuperMix UDG (Catalog no. 11730-017) is a 2X-concentrated, ready-to-use mixture containing all components except primers and template. It uses Platinum^*81 Taq DNA polymerase and has been specifically formulated to provide optimal performance in real-time PCR systems. Continued on next page

Real-Time PCR, continued

Instrument LUX™ Primers are compatible with a wide variety of real-time PCR Specifications instruments with various detection capabilities. See page 2 for a partial list of compatible instruments. A protocol for instruments that use PCR tubes/plates is provided on page 9, A protocol for the LightCycler^® is provided on page 10. At a ininimum, the instrument used to perform real-time PCR with LUX™. • Detect fluorescence at each PCR cycle • Excite and detect RAM-labeled LUX™ Primers near their excitation/emission wavelengths of 490/520 nm, and/ or • Excite and detect JOE-labeled LUX™ Primers near their excitation/emission wavelengths of 520/550 nm Please refer to the specific instrument's user manual for operating instructions.

Instrument Please follow the manufacturer's instructions for configuring your real-time Settings PCR instrument for use with LUX™ Primers. Note the following settings: • LUX™ Primers are compatible with standard melting curve analysis, if your instrument allows that option. Program your instrument accordingly. • The quencher setting on the instrument should reflect the fact that LUX™ Primers do not contain a quencher. • We recommend the use of ROX Reference Dye (Cat. no. 12223-023) for normalization of well-to-well variation with instruments that are compatible with this option. Adjust your instrument settings accordingly. Continued on next page

Real-Time PCR, Continued

Protocol for The following protocol uses Platinum¹⁸ Quantitative PCR SuperMix-UDG with Instruments Using ROX reference reagent. It has been optimized for use with real-time PCR PCR Tubes or instruments that use PCR tubes or plates. A protocol for the Roche Plates LightCycler^® is provided on the following page. Mote: The following protocol uses a 50-μl reaction volume; smaller volumes may be used, depending on the requirements of your instrument. Before proceeding, see the real-time PCR guidelines on the previous pages. For multiplex reactions, see the guidelines on page 11. 1. To reduce well-to-well variation, prepare a Master Mix of all the reaction ingredients except template. The following table provides Master Mix volumes or one reaction and 50 reactions (scale up or down as needed): Component Vol/l rxn Vol/50 rxns Platinum^® Quantitative PCR SuperMix-UDG¹ 25 μl 1250 μl ROX Reference Dye l μl 50 μl Labeled LUX™ Primer (10 μM) l μl 50 μl Unlabeled primer (10 μM) l μl 50 μl Sterile distilled water² to 40 μl to 2000 μl '60 U/ml Platinum^® Taq DNA polymerase, 40 mM Tris-HCI (pH 8.4), 100 mM KCL 6 mM MgCb, 400 μM dGTP, 400 μM dATP, 400 μM dCTP, 800 μM dUTP, 40 U/ml UDG, and stabilizers. ^""or use DNase/RNase Free Distilled Water (Cat. No.10977-015). 2. Program the real-time PCR instrument as follows: 3-Steρ Cycling (recommended) 2-Step Cycling (optional) 50°C, 2 min hold (UDG treatment) 50°C, 2 min hold (UDG treatment) 95°C, 2minhold 95°C, 2 minhold 45 cycles of: 45 cycles of: 95°C, 15 s 95°C, 15 s 55°C,30 s 60-65°C, 30-45 s 72°C,30 s Melting Curve Analysis (optional) Refer to instrument documentation Add 40 μl of the Master Mix to an optical PCR tube or each well of a 96-well PCR plate. 4. Add 10 μl of template diluted in TE or sterile dH₂0 to the tube or each well of the 96-well PCR plate. Cap or seal the tube/plate. 5. Gently mix and make sure that all components are at the bottom of the tube/plate wells. Centrifuge briefly if needed. Place reaction in the real-time PCR instrument and run the program. Collect and analyze results. Real-Time PCR, Continued

Protocol for the Hie following protocol uses Platinum^® Quantitative PCR SuperMix-UDG and

Roche LightCycler^® has been optimized for the Roche LightCycler^®. Consult the LightCycler^® documentation for detailed instructions on preparing the capillary tubes and operating the instrument. FAM-labeled LUX™ Primers are also compatible with Roche enzyme mixes. Note JOE-labeled LUX™ Primers are not compatible with the current version of the LightCycler^®; use FAM-labeled primers only. The following protocol uses a 20-μl reaction volume. Before proceeding, see the real-time PCR guidelines on the previous pages. 1. To reduce well-to-Well variation, prepare a Master Mix of all the reaction ingredients except template. The following table provides volumes for one reaction and 34 reactions (scale as needed): Component Vol/l rxn Vol/34 rxns Platinum^® Quantitative PCR SuperMix-UDG¹ 10 μl 340 μl FAM-labeled LUX™ Primer (10 μM) 1 μl 34 μl Unlabeled primer (10 μM) l μl 34 μl Bovine serum albumin (5 mg/ml)² l μl 34 μl Platinum^® Taq DNA Polymerase³ 0.12 μl 4 μl Sterile distilled water* to 18 μl to 612 μl «60 U/ml Platinum^® Taq DNA polymerase, 40 mM Tris-HCI (pH 8.4), 100 mM KC1, 6 mM MgCh, 400 μM dGTP, 400 μM dATP, 400 μM dCTP, 800 μM dUTP, 40 U/ml UDG, and stabilizers. ^''Validated with nαn-acetylated Ultrapure BSA (10% solution) from Panvera (Cat. nos. P248 andP2046). ³Total units of Platinum^® Taq DNA Polymerase in the reaction is 1.2 (including 0.6 U in Platinum^® Quantitative PCR SuperMix-UDG *or use DNase/RNase Free Distilled Water (Cat. No.10977-015). 2. Set the fluorescence on the Roche LightCycler^® to the Fl channel. 3. Program the instrument as follows: Thermal Cycling Melting Curve Analysis (optional) Program choice: Amplification Program choice: Melting curve Analysis mode: Quantification Analysis made: Melting curves Cycling: Cycling: 50°C_/ 2 min hold (UDG treatment) 95°C, 0s 95°C_/2 i hold 55°C, 15 sec 45 cycles of: 95°C, 0 (increase 0.1°C/s with 94°C, 5 s continuous acquisition) 55°C, 10 s (single acquire) 40°C, 0 s 72°C, 10 s 4. Add 18 μl of Master Mix to each capillary tube of the LightCycler^®. 5. Add 2 μl of template to each tube, and cap the tube. 6. Centrifuge the tubes at 700 x g for 5 seconds. 7. Place the reaction tubes in the rotor of the LightCycler^® and run the program. Collect and analyze results. Multiplex Real-Time PCR

Multiplex In multiplex real-time PCR, different sets of primers with different labels are Real-Time PCR used to amplify separate genes on the template DNA. Multiplexing with LUX™ Primers offers simplified kinetics when compared with probe-based technologies, because only two oligos are used per target. LUX™ Primers have been tested in multiplex reactions using a FAM-labeled primer set for the gene of interest and a JOE-labeled set for a housekeeping gene used as an internal control to normalize between different reactions. We recommend using Certified LUX™ Primer Sets for Housekeeping Genes for the internal control. In a standard multiplex reaction, you can include the additional primers at the same volumes and concentration as the primers in a singleplex reaction, as shown in the example mixture below: Component Volume Platinum^® Quantitative PCR SuperMix-UDG (2X) 25 μl ROX Reference Dye (50X) l μl Template 10 μl Forward primer 1 (FAM label) (10 μM) l μl Reverse primer 1 (10 μM) l μl Forward primer 2 (JOE label) (10 μM) l μl Reverse primer 2 (10 μM) l μl Sterile distilled water to 50 μl Reduce the volume of water to compensate for the additional primer volume. All other reaction volumes remain the same. Follow the thermal cycling guidelines provided in Protocol for Instruments Using PCR Tubes or Plates on page 9. If you have difficulty performing the multiplex reaction using these guidelines, see the optimization hints below.-

Optimizing If you notice a decline in real-time PCR efficiency in your multiplex real-time

Multiplex PCR, you can optimize the reaction by performing the steps listed below.

Conditions Note: We recommend that you perform one optimization step and then repeat the reaction to test for efficiency before moving on to the next step: 1. Reduce the primer concentration of the gene with the highest abundance (typically the housekeeping gene) to 1/4 the primer concentration of the other gene. For example, in a standard 50 μl reaction, you would add the primers for the less abundant gene at 1 μl each, and add the primers for the more abundant gene at 0.25 μl each. 2. Increase the MgCk in the reaction from 3 mM to 5 mM. 3. Double the amount of polymerase enzyme (to 0.06 U per μl of reaction volume). If you are using Platinum^® Quantitative PCR SuperMix-UDG, add Platinum^® Taq DNA polymerase stand-alone enzyme (Catalog no. 10966-018) to double the amount of enzyme. 4. Increase the dNTP concentrations in the reaction to 400 μM each. Two-Step Real-Time RT-PCR

Introduction For real-time RT-PCR applications, we recommend a two-step protocol so that the RT and PCR modules can be optimized separately for maximum efficiency and specificity. This section provides an optimized protocol for performing reverse transcription as part of a two-step real-time RT-PCR protocol. You can use the resulting cDNA in the real-time PCR reaction on pages 7-10.

Template The target template for real-time RT-PCR is RNA— usually total cellular RNA Specifications or mRNA. The amount of RNA typically varies from 1 pg to 100 ng of template per assay. The purity and integrity of the RNA have a direct impact on results. RNase and genomic DNA contamination are the most common problems, and purification methods should include RNase inhibitors and DNase digestion to minimize these. We recommend using the Micro-to-Midi Total RNA Purification System (Catalog no. 12183-018) or TRIzol^® reagent (Catalog no. 15596-026) to isolate total RNA. High-quality total RNA can be purified from as little as 100 cells up to 10⁷ cells or 200 mg of tissue. To isolate mRNA, we recommend using the FastTrack^® 2.0 mRNA Isolation Kit (Catalog no. K1593-02).

Enzyme We recommend using Superscript™ II or Superscript™ HI RT for the reverse Specifications transcription reaction. The sample protocol on page 13 uses the Superscript™ First-Strand Synthesis System for RT-PCR (Catalog no.11904-018), available from Invitrogen, which includes all components needed for the first-strand synthesis reaction except the RNA.

Removing We recommend that you decrease the genomic DNA content in the RNA Genomic DNA from sample by performing a digest with DNase I, Amplification Grade (Catalog RNA Samples no.18068-015), as described below. The DNase I digest is designed for up to 1 μg of RNA; for larger amounts of RNA, increase volumes accordingly. Combine the following in a tube on ice: Component Cone. ^"Volume RNA template — x μl DNase reaction buffer 10X l μl DNase I, Amplification Grade l U/μl l μl DEPC-treated ddHaO to lO μl 1. Incubate at room temperature for 15 min. 2. Add 1 μl of 25-mM EDTA solution to the reaction mixture and incubate at 65°C for 10 min to inactivate the DNase I. Continued on next page Two-Step Real-Time RT-PCR, continued

Reverse The following protocol can be used with either the Superscript™ First-Strand

Transcription Synthesis System for RT-PCR (with Superscript™ π RT) or the Superscript™ DJ

Protocol First-Strand Synthesis System for RT-PCR (with Superscript™ HI RT). The protocol has been optimized for LUX™ Primers. Follow this protocol to generate cDNA, which can then be used in real-time PCR (see pages 7-10). 1. Combine the following kit components in a tube on ice. For multiplex reactions, a master mix without RNA may be prepared: 01igo(dT)i2-i8 (0.5 μg/μl) or 01igo(dT)2o (50 M)* 0.5 μl Random hexamers (50 ng/μl) 0.5 μl RNA (up to 1 μg) x μl lOx Buffer 2 μl 25 m MgCl2 4 μl lOmM dNTP i μl 0.1 M DTT 2 μl RNaseOUT™ (40 U/μl) l μl Superscript™ II RT (50 U/μl) or Superscript™ HI RT (200 U/μl) l μl DEPC-treated ddH-O to 20 μl *01igo(dT)ιι-u is recommended for use with Superscript^*"' π RT; oligo(dT)2o is recommended for use with Superscript'^"' Id RT 2. Incubate tube at 25°C for 10 min. 3. Incubate tube at 42°C for 50 min. 4. Terminate the reaction at 70°C at 20 min, and then chill on ice. 5. Add 1 μl (2 U) of E. cόli RNaseH and incubate at 37°C for 20 min. Store the reaction at -20°C until use. Use 2-8 μl of cDNA for real-time PCR, as described on pages 7-10.

One-Step Real-Time RT-PCR

Introduction This section provides information and a protocol for performing one-step realtime RT-PCR using LUX™ Primers. One-step RT-PCR is a complex reaction in which both reverse transcription and PCR are carried out in the same tube. The one-step reaction described in this section uses the Superscript™ HI Platinum^® One-Step Quantitative RT-PCR System for superior specificity and sensitivity with LUX™ Primers.

Primer For optimal PCR, primer titrations of 50-500 nM per primer are recommended. Concentration The 50-μl sample reaction on page 16 uses 200 nM of each primer, equivalent to 1 μl of a 10 μM primer solution. See also the Important note below.

In one-step RT-PCR, the reverse primer drives the reverse transcription reaction. We have found that doubling the concentration of the reverse primer from 200 nM to 400 nM can in some cases decrease the cycle threshold for detecting a given target concentration, and thus increase sensitivity. See

pages 3-4 for guidance on primer design.

Template The target template for one-step real-time RT-PCR is RNA — sually total Specifications cellular RNA or mRNA. The amount of template typically ranges from 1 pg to 100 ng per assay. The purity and integrity of the RNA have a direct impact on results. RNase and genomic DNA contamination are the most common problems, and purification methods should be designed to avoid these. We recommend using the Micro-to-Midi Total RNA Purification System (Catalog no 12183-018) or TRIzol^® reagent (Catalog no.15596-026) to isolate total RNA. High-quality total RNA can be purified from as little as 100 cells up to 10⁷ cells or 200 mg of tissue. To isolate mRNA, we recommend using the FastTrack^® 2.0 mRNA Isolation Kit (Catalog no. K1593-02).

Enzyme The one-step RT-PCR enzyme mix should be optimized for real-time PCR. We Specifications recommend using the Superscript™ HI Platinum^® One-Step Quantitative RT- PCR System (Catalog nos.11732-020 and -088), which uses a Superscript™ HI RT/Platinum^® Taq enzyme mix. It has been optimized for use in real-time fluorescent PCR systems. See the sample reactions on pages 16-17.

Magnesium The optimal MgCl. concentration for a given target/primer /polymerase Concentration combination can vary between 1 mM and 10 mM, but is usually in the range of 3 mM (see the sample reaction on page 16).

Continued on next page One-Step Real-Time RT-PCR, Continued

Instrument LUX™ Primers are compatible with a wide variety of real-time PCR Specifications instruments with various detection capabilities. See page 2 for a partial list of compatible instruments. A one-step real-time RT-PCR protocol for instruments that use PCR tubes/plates is provided on page 16. A protocol for the LightCycler^® is provided on page 17. At a minimum, the instrument used to perform one-step real-time RT-PCR with LUX™ Primers must be able to: » Detect fluorescence at each PCR cycle • Excite and detect FAM-labeled LUX™ Primers near their excitation/emission wavelengths of 490/520 nm, and/or • Excite and detect JOE-labeled LUX™ Primers near their excitation/emission wavelengths of 520/550 nm

Instrument Please follow the manufacturer's instructions for configuring your real-time Settings PCR instrument for use with LUX™ Primers. Note the foHowing settings: • LUX™ Primers are compatible with standard melting curve analysis, if your instrument allows that option. Program your instrument accordingly. • The quencher setting on the instrument should reflect the fact that LUX™ Primers do not contain a quencher. • We recommend the use of ROX Reference Dye (Catalog no.12223-023) for normalization of well-to-weU variation with instruments that are compatible with this option. Adjust your instrument settings accordingly. • Program the instrument to perform cDNA synthesis immediately followed by PCR amplification.

Removing We recommend that you decrease the genomic DNA content in the RNA

Genomic DNA from sample by performing a digest with DNase I, Amplification Grade (Catalog RNA Samples ^no- 18068-015), as described below. The DNase I digest is designed for up to 1 μg of RNA; for larger amounts of RNA, increase volumes accordingly. Combine the following in a tube on ice: Component Cone. Volume RNA template κ μl DNase reaction buffer 10X I μl DNase I, Amplification Grade l U/μl l μl DEPC-treated ddH₂0 to lO μl 1. Incubate at room temperature for 15 min. 2. Add 1 μl of 25-mM EDTA solution to the reaction mixture and incubate at 65°C for 10 min to inactivate the DNase I. To verify the absence of genomic DNA in the RNA sample, prepare a control reaction identical to the reactions on pages 16-17, using 2 U of Platinum^® Taq DNA polymerase (Catalog no.10966-018) in place of the Superscript™ HI RT/Platinum^® Taq Mix.

Continued on next page One-Step Real-Time RT-PCR, continued

Protocol for The following protocol using the Superscript™ HI Platinum^® One-Step Instruments Using Quantitative RT-PCR System has been optimized for LUX™ Primers. Further PCR Tubes or optimization may be required. Plates Note: Keep all components, reaction mixes and samples on ice. After assembly, transfer the reaction to a thermal cycler preheated to the cDNA synthesis temperature and immediately begin RT-PCR. We recommend performing the cDNA synthesis reaction at 50°C, but higher temperatures (up to 60°C) may be required for high GC content templates. RNase inhibitor proteins, such as RNaseOUT™ (Catalog no. 10777-019), may be added to the reaction to safeguard against degradation of RNA. 1. The following table provides Master Mix volumes for a standard 50-μl reaction size. Note that preparation of a master mix is crucial in quantitative applications to reduce pipetting errors. Component Vol/lrxn VoltlOQ rxns Superscript™ HI RT/Platinum^® Taq Mix l μl 100 μl 2X Reaction Mix¹ 25 μl 250O μl ROX Reference Dye (optional) l μl 100 μl Labeled LUX™ Primer (10 μM) l μl 100 μl Unlabeled primer (10 μM)² l μl 100 μl RNaseOUT™ (optional) l μl 100 μl Sterile distilled water to 40 μl to 4000 μl "Supplied at 2X concentration: includes 0.4 mM of each dMTP and 6 mM MgSO "Seethe Important note on primer concentration on page 14. 2. Progra the instrument with the following thermal cycling protocol (for cDNA synthesis, use a 15-min incubation at 50°C as a starring point): cDNA synthesis: , 50°C for l5 inhold PCR; 95°C for 2 min hold 40-50 cycles of: 95°C, 15 s 60°C,30s Melting Curve Analysis (optional) Program according to instrument instructions 3. For each reaction, add 40 μl of the master mix to a 0.2-ml microcentrifuge tube or each weU of a 96-well PCR plate on ice. 4. Add 10 μl of sample RNA (1 pg to 1 μg total RNA) to each tube/plate weU, and cap or seal. Gently mix and make sure that all components are at the bottom of the tube/plate wells. Centrifuge briefly if needed. Place reactions in a preheated thermal cycler programmed as described above. Collect data and analyze results.

Continued on next page One-Step Real-Time RT-PCR, Continued

Protocol for the The following protocol using the Superscript™ HI Platinum^® One-Step

Roche LightCycler^® Quantitative RT-PCR System has been optimized for LUX™ Primers and the Roche LightCycler^®. Further optimization may be required. FAM-labeled ' LUX™ Primers are also compatible with Roche enzyme mixes. Note: JOE-labeled primers are not compatible with the current version of the LightCycler^®; use FAM-labeled primers only. After assembly, transfer the reaction to a thermal cycler preheated to the cDNA synthesis temperature and immediately begin RT-PCR. We recommend performing the cDNA synthesis reaction at 50°C, but higher temperatures (up to 60°C) may be required for high GC content templates. RNase inhibitor proteins, such as RNaseOUT™ (Catalog no.10777-019), maybe added to the reaction to safeguard against degradation of RNA. 1. The following table provides Master Mix volumes for a standard 20-μl reaction size. Note that preparation of a master mix is crucial in quantitative applications to reduce pipetting errors. Component Vol/l rxn VolV3 rxns Superscript™ HI RT/Platinum^® Taq Mix 0.8 μl 27.2 μl 2X Reaction Mix¹ 10 μl 340 μl FAM-labeled LUX™ Primer (10 μM)² l μl 34 μl Unlabeled primer (10 μM)³ l μl 34 μl Bovine serum albumin (5 mg/ml)⁴ l μl 34 μl Sterile distilled water to lβ μl to 612 μl 'Includes 0.4 mM of each dNTP and 6 mM MgS0₄ ^""In the LightCycler^® reaction, the LUX™ Fluorogenic Primer must be FAM labeled. ^e the Important note on primer concentration on page 14. Validated with non-acetylated ^"Ultrapure BSA (10% solution) from Panvera (Cat. nos.P2489 nd P2046) 2. Set the fluorescence on the Roche LightCycler^® to the Fl channel. 3. Program the instrument as follows: Thermal Cycling Melting Curve Analysis (optional) Program choice: Amplification Program choice: Melting curve Analysis mode: Quantification Analysis mode: Melting curves Cycling: Cycling: 45°C_/ 30 min hold (cDNA synthesis) 95°C, 0 s 95°C,2minhold 55°C, 15 sec 50 cycles of: 95°C,0 (increase 0.1°C/s with 95°C, 5 s continuous acquisition) 55°C, 10 s (single acquire) 40°C, 0 s 72°C, 10s 4. Add 18 μl of Master Mix to each capillary tube of the LightCycler^® on ice. 5. Add 2 μl of sample RNA (1 pg to 1 μg total RNA) to each capillary tube and cap the tube. 6. Centrifuge the tubes at 700 x g for 5 seconds. 7. Place the reaction tubes in the rotor of the LightCycler^® and run the program. Collect and analyze results. Troubleshooting

Problem Cause Solution

Signal in controls with no DNA contamination Ensure that amplification reactions are assembled template in a DNA-free environment. Use of aerosol- resistant barrier tips is recommended. Take care to avoid cross-contamination between primers or template DNA in different reactions. Run PCR product on an agarose gel in an area separate from the reaction assembly area to confirm product. Amplification of PCR Analyze PCR product on an agarose gel in an area carryover products separate from the reaction assembly area. Use Platinum^® Quantitative PCR SuperMix-UDG as specified in the sample protocols on pages 9- 10. Since dUTP is substituted for dTTP in the reaction cocktail, any amplified DNA will contain uracil. UDG prevents reampUfication of PCR carryover products by removing uracil residues from single or double stranded DNA. dU- containing DNA that has been digested with UDG is unable to serve as template in future PCRs. UDG is inactivated at high temperature during PCR thermal cycling, thereby allowing amplification of genuine target sequence(s). Primer dimers Perform melting curve analysis of the PCR product; identify dimers by lower melting point temperature. Confirm that primer designs have low scores (0.0-4.0) to nunimize self-annealing. Redesign primers if necessary. When redesigning primers, note that you can first try redesigning only the unlabeled primer to save the cost of the LUX™ primer.

No or low signal Instruments setting not Confirm that the cycling parameters are correct, optimal the quencher is set to none, and the reference dye setting is correct. Primer/template sequences do Confirm that the sequences match, not match Primer designs are not optimal Confirm that the primer design scores are within the 0.0-4.0 range and the optimal melting temperatures have been specified. Redesign primers if necessary. When redesigning primers, note that you can first try redesigning only the unlabeled primer to save the cost of the LUX"¹ primer.

Poor standard curve and Reaction is not optimized Reoptimize reaction conditions. Prepare primer dynamic range titrations if necessary. Reference dye not used Use ROX Reference Dye as specified. Accessory Products

Products The following products are available for use with LUX™ Primers in real-time PCR and RT-PCR protocols:

Purchaser Notification

Limited Use Label The purchase of this product conveys to the buyer the non-transferable right to use the License No. 114: purchased amount of the product and components of the product in research LUX™ Fluorogenic conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) Primer materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for commercial purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for the conunercial purposes of the buyer, and that such collaborator agrees in writing (a) to not transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for commercial purposes. Commercial purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned by Invitrogen based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manuf acture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the products with a full refund. For information on purchasing a license to this product for purposes other than research, contact Licensing Department, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500.

Limited Use Label This product is optimized for use in the Polymerase Chain Reaction (PCR) covered by License No.4: patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche, Ltd. Products for PCR ("Roche"). No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser by the purchase of this product A license to use the which do not PCR process for certain research and development activities accompanies the purchase include any rights of certain reagents from licensed suppliers such as Invitrogen, when used in to perform PCR conjunction with an Authorized Thermal Cycler, or is available from Applied Biosystems. Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404 or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501. Technical Service

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References

Ailenberg, M., and Silverman, M. (2000) Controlled hot start and improved specificity in carrying out PCR utilizing touch-up and loop incorporated primers (TULIPS). BioTechniques 29, 1018-1024. Bustin, S. A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol.25, 169-193. Cardullo, R. A., Agrawal, S., Flores, C, Zamecnik, P. C, and Wolf, D. E. (1988) Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc. Natl Acad. Sci. USA 85, 8790-8794. Crockett, A.O., and Wittwer, C.T. (2001) Fluorescein-labeled oligonucleotides for real-time per: using the inherent quenching of deoxyguanosine nucleotides. A al. Biochem.290, 89-97. Higuchi, R., Fockler, C, Wabh, P.S., and Griffith, R. (1992) Simultaneous amplification and detection of specific DNA sequences. Biotechnology 10, 413-417. Higuchi, R., Fockler, C, Dollinger, G., and Watson, R. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11, 1026-1030. Holland et al. (1991) Detection of specific polymerase chain reaction product by utilizing the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sd. USA 88, 7276- 7280. Kaboev, O. K., Luchkina, L. A., Tret'iakov, A. N-, and Bahrmand, A.R. (2000) PCR hot start using primers with the structure of molecular beacons (hairpin-like structure). Nucleic Acids Res.28, e94. Knemeyer, J.P., Marme, N., and Sauer, M. (2000) Probes for detection of specific DNA sequences at the single-molecule level. Anal. Chem. 72,3717-3724. Murchie, A. I. H., Clegg, R. M., vonKitzing, E., Duckkett, D. R., Didkmann, S., and Lilley D. M. J. (1989) Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules. Nature Ml, 763-766. Myakishev, M. V., Khrip in, Y., Hu, S., and Hamer, D. H. (2001) High-throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers. Genottle Res.11, 163-169. Nazarenko, I., Lowe, B., Darfler, M., Ikonomi, P., Schuster, D., and Rashtchian, A. (2002) Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore. NucL Acids Res. 30, e37 Nazarenko, I., Pires, R._f Lowe, B., Obaidy, M., and Rashtchian, A. (2002) Effect of prhnary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes. Nuc . Acids Res.30, 2089-2095 Nazarenko, I.A., Bhatnagar, S.K., and Hohman, R.J. (1997) A closed tube format for amplification and detection of DNA based on energy transfer. Nucleic Acids Res. 25, 2516-2521. Nuovo, G. J., Hohman, R. J., Nardone, G. A., and Nazarenko I. (1999) In situ amplification using universal energy transfer-labeled primers. /. Histochem. Cytockem. 47, 273-279. Todd, A. V., Fuery, C. J., I pey, H. L., Applegate, T. L. and Haughton, M.A. (2000) DzyNA-PCR: use of DNAzymes to detect and quantify nucleic acid sequences in a real-time fluorescent format Clin. Chem. 46, 625-630. Tyagi, 5., and Kramer, F.R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnol. 14, 303-^08. Wittwer, C.T., Herrmann, M.G., Moss, A.A., Rasmussen, R.P. (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. BioTechniques 22, 130-138. ©2002-2003 Invitrogen Corporation. AH rights reserved.

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Claims

What is claimed is:

1. An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, the assay being a selective ligation and amplification method of the type using a controlled-temperature reaction mixture including the target sequence, ligatable first and second primers having at least a portion substantially complementary to first and second segments of the target sequence, respectively, and a third primer that is substantially complementary to a random sequence segment of the first and second primers, wherein the improvement comprises: homogeneously detecting amplified target sequence using a dye specific for binding to double-stranded (ds) DNA that fluoresces upon binding target sequence.

2. An improved assay according to claim 1 wherein a nucleotide complementary to the SNP of the target sequence is present at the 5 '-end of the second primer.

3. An improved assay according to claim 1, wherein the. dye comprises SYBR^® Green.

4. An improved assay according to claim 1, wherein the assay further comprises: using a first primer and a second primer at concentrations such that a ligated product produces exponentially amplified target sequence detectable above linearly amplified non-ligated primer product.

5. An improved assay according to claim 1, wherein the assay further comprises: using a plurality of first primers and second primers designed to generate amplified target sequences with differential melting curves; distinguishing individual amplified target sequences by differences in melting temperatures (T_ms).

6. An improved assay according to claim 1, wherein the first and second primers contain degenerate base-pairing positions to allow hybridization to variable regions in target sequences adjacent to the SNP.

7. An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, the assay being a selective ligation and amplification method of the type using a temperature-controllable reaction mixture including the target sequence, ligatable first and second primers having at least a portion substantially complementary to first and second segments of the target sequence, respectively, and a third primer that is substantially complementary to a random sequence segment of the first and second primers, wherein the improvement comprises:

^' - - ^• - detecting amplified target sequence using a probe specific for hybridizing across a ligation junction formed between the first primer and second primer after binding to the target sequence wherein the probe specific for hybridizing across the ligation junction contains a molecular beacon.

8. An improved assay according to claim 7, wherein the probe specific for hybridizing across the ligation junction has a fluorescent group and a fluorescence-modifying group.

9. An improved assay according to claim 8, wherein the fluorescent group is quenched when the probe is not bound across the ligation junction and the fluorescent group fluoresces when the probe is bound across the ligation junction.

10. An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, the assay being a selective ligation and amplification method of the type using a temperature-controllable reaction mixture including the target sequence, ligatable first and second primers having at least a portion substantially complementary to first and second segments of the target sequence, respectively, and a third primer that is substantially complementary to a random sequence segment of the first and second primers, wherein the improvement comprises: detecting amplified target sequence using a probe specific for hybridizing to a region of the target sequence wherein the probe contains a fluorescent group and a fluorescence-modifying group.

11. An improved assay according to claim 10, wherein upon extension of the probe, the fluorescence-modifying group is excised and the fluorescent group fluoresces.

12. An improved assay according to claim 7 or 10, wherein the fluorescent group is quenched before incorporation into double-stranded product and is dequenched after incorporation into double-stranded product.

13. An improved assay according to claim 12, wherein the fluorescent group is quenched by secondary structure before incorporation into double-stranded product, such that before incorporation, a sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group, quenching the fluorescent group.

14. A nanoliter sampling array comprising: a) a first platen having at least one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes; wherein each through-hole contains i) a first primer having at least a portion of its 3 '-end substantially complementary to a first segment at a first end of a potential nucleic acid target sequence; and ii) a second primer having at least a portion of its 5'-end substantially complementary to a second segment at a second end of the potential nucleic acid target sequence, the first and second primers being ligatable upon binding to the potential nucleic acid target sequence.

15. A nanoliter sampling array according to claim 14, further comprising: a second platen having at least one hydrophobic surface and having a high- density microfluidic array of hydrophilic through-holes; wherein the first and second platen are fixedly coupled such that the through-holes of each are aligned.

16. A nanoliter sampling array according to claim 14, wherein at least one pair of aligned through-holes contains first reagents for a first assay process and second reagents for a second assay process.

17. An array according to claim 16, wherein one of the assay processes is PCR amplification.

18. An array according to claim 16, wherein one of the assay processes is detection of amplified target nucleic acid sequence having a SNP.

19. An array according to claim 18, wherein detection of amplified target nucleic acid sequence comprises using a dye specific for binding to double-stranded (ds) DNA that fluoresces upon binding target sequence.

20. An array according to claim 18, wherein detection of amplified target nucleic acid sequence comprises distinguishing individual amplified target sequences by differences in melting temperatures (T_ms).

21. An array according to claim 18, wherein detection of amplified target nucleic acid sequence comprises using a probe specific for hybridizing across a ligation junction formed between the first primer and second primer after binding to the target sequence, wherein the probe has a fluorescent group and a fluorescence-modifying group.

22. An array according to claim 18, wherein detection of amplified target nucleic acid sequence comprises using a probe containing a fluorescent group and a fluorescence- modifying group specific for hybridizing to a region of the target sequence wherein upon extension of the probe, the fluorescence-modifying group is excised and the fluorescent group fluoresces.

23. An array according to claim 22, wherein the fluorescent group is quenched before incorporation into double-strand product and is dequenched after incorporation into double-stranded product.

24. An array according to claim 23, wherein the fluorescent group is quenched by secondary structure before incorporation into double-stranded product such that before incorporation, a sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group, quenching the fluorescent group.

25. A nanoliter sampling array according to any of claims 14-24, wherein the primers are affixed on, within or under a coating of the sample through-holes by drying, the coating comprising a biocompatible material.

26. A method of identifying a SNP in a target sequence of nucleic acid, the method comprising: •providing a first sample platen having a high-density microfluidic array of through-holes, each through-hole having a first primer having at least a portion substantially complementary to a first segment of the target sequence, a second primer having at least a portion substantially complementary to a second segment of the target sequence, the 5'-end of the second primer ligatable to the 3'-end of the first primer after binding nucleic acid target sequence, and a third primer that is substantially complementary to a random sequence segment of the first and second primers; introducing a sample containing a target sequence of nucleic acid having a SNP of interest to the through-holes in the array; introducing reagents to the through-holes in the array, the reagents including a reagent for effecting amplification, a reagent for effecting ligation, and at least four different nucleotide bases; effecting ligation of the first and second primers to produce a ligated product; effecting amplification of the ligated product and target sequence; detecting amplified target sequence.

27. A method of identifying a SNP in a target sequence of nucleic acid according to claim 26, wherein effecting ligation and effecting amplification comprises addition of a ligase and a polymerase followed by subjecting the array to controlled-temperature conditions.

28. A method according to claim 26 wherein detecting comprises using a dye specific for binding to double-stranded (ds) DNA that fluoresces upon binding target sequence.

29. A method according to claim 26 wherein detecting comprises distinguishing individual amplified target sequences by differences in melting temperatures (T_ras).

30. A method according to claim 26 wherein detecting comprises using a probe specific for hybridizing across a ligation junction formed between the first primer and second primer after binding to the target sequence, wherein the probe has a fluorescent group and a fluorescence-modifying group.

31. A method according to claim 26 wherein detecting comprises using a probe containing a fluorescent group and a fluorescence-modifying group specific for hybridizing to a region of the target sequence wherein upon extension of the probe, the fluorescence-modifying group is excised and the fluorescent group fluoresces.

32. An improved assay according to claim 30, wherein the fluorescent group is quenched before incorporation into double-strand product and is dequenched after incorporation into double-stranded product.

33. A method according to claim 32, wherein the fluorescent group is quenched by secondary structure before incorporation into double-stranded product, such that before incorporation, a sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group, quenching the fluorescent group.

34. A kit for use in identification of amplified target nucleic acid sequences, the kit comprising: a) a sample platen having one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes; wherein each sample platen through-hole contains at least i) a first primer having at least a portion substantially complementary to a first segment of potential nucleic acid target sequence; ii) a second primer having at least a portion substantially complementary to a second segment of the potential nucleic acid target sequence, the first and second primers ligatable upon binding to the potential nucleic acid target sequence; b) a reagent platen having a high-density microfluidic array of through-holes, each reagent platen through-hole containing at least i) a third primer that is substantially complementary to a random sequence segment of the first and second primers; ii) at least four different nucleotide bases; iii) a reagent for effecting ligation; and iv) a fluorescent dye the reagent platen having a structural geometry that corresponds to the sample platen allowing delivery of reagent components and target nucleic acid sample to the primers in the sample platen.

35. A kit for use in identification of amplified target nucleic acid sequences according to claim 34, wherein a PCR-compatible buffer is also included.

36. A kit according to claim 34, wherein the fluorescent dye comprises SYBR^® Green I, SYBR^® Green II, YOYO^®-l, TOTO^®-l, POPO^®-3, or ethidium bromide.

37. A kit according to any of claims 34-36, wherein the primers are affixed on, within or under a coating of the sample through-holes by drying, the coating comprising a biocompatible material.

38. An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, the assay being a selective ligation and amplification method of the type using a controlled- temperature reaction mixture including the target sequence, ligatable first and second primers having at least a portion substantially complementary to first and second segments of the target sequence, respectively, and a third primer that is substantially complementary to a random sequence segment of the first and second primers, wherein the improvement comprises: detecting one or more amplified target sequences in a single-tube reaction system using one or more probes specific for hybridizing to a region of one or more target nucleic acid sequences, wherein the one or more probes each contain a distinct fluorescent group and a fluorescence-modifying group and wherein hybridization of the one or more probes results in fluorescence of the distinct fluorescent group.

39. An improved assay according to claim 38, wherein upon extension of the probe, the •fluorescence-modifying .group is .excised and the fluorescent group fluoresces.

40. An improved assay according to claim 38, wherein the fluorescent group is quenched before incorporation into double-strand product and is dequenched after incorporation into double-stranded product.

41. An improved assay according to claim 40, wherein the fluorescent group is quenched by secondary structure before incorporation into double-stranded product, such that before incorporation, a sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group, quenching the fluorescent group.

42. An improved assay according to claim 38, wherein the one or more target nucleic acid sequences is 2, each having a distinct SNP of interest.

43. An improved assay according to claim 42, wherein the one or more hybridizable probes is 2, each having a distinct fluorophore and unique sequence that hybridizes to and amplifies each of the 2 target nucleic acid sequences.

44. An improved assay according to claim 38, wherein the one or more target nucleic acid sequences is 3, each having a distinct SNP of interest.

45. An improved assay according to claim 44, wherein the one or more hybridizable probes is 3, each having a distinct fluorophore and unique sequence that hybridizes to and amplifies each of the 3 target nucleic acid sequences.

46. An improved assay according to claim 38, wherein the one or more target nucleic acid sequences is 4, each having a distinct SNP of interest.

47. An improved assay according to claim 46, wherein the one or more hybridizable probes is 4, each having a distinct fluorophore and unique sequence that hybridizes to and amplifies each of the 4 target nucleic acid sequences.

48. An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, the assay being a selective ligation and amplification method of the type using a controlled- temperature reaction mixture including the target sequence, ligatable first and second primers having at least a portion substantially complementary to first and second segments of the target sequence, respectively, and a third primer that is substantially complementary to a random sequence segment of the first and second primers, wherein the improvement comprises: detecting one or more amplified target sequences in a single-tube reaction system using one or more fourth primers, each having a fluorescent group and a fluorescent- modifying group, and each being complementary to a unique region of a ligated template for the one or more target nucleic acid sequences, wherein upon fourth primer incorporation into and amplification of the one or more target nucleic acid sequences, fluorescence of the distinct fluorescent group occurs such that detection of one or more amplified target nucleic acid sequences in a single-tube reaction system results.

49. An improved assay according to any of claims 1, 7, 10, 26, or 48, further comprising using a polymerase that lacks 5' to 3' exonuclease activity.

50. An improved assay according to any of claims 1, 7, 10, 26, or 48, further comprising using a polymerase that lacks 3' to 5' exonuclease activity.

51. An improved assay according to any of claims 1, 7, 10, 26, or 48, further comprising using a polymerase that lacks 5' to 3' exonuclease activity and 3' to 5' exonuclease activity.

52. An improved assay according to any of claims 7, 10, 38 or 48, wherein the distinct fluorescent groups comprise Redmond Red™, Yakima Yellow™, and the fluorescence-modifying group comprises an Eclipse™ non-fluorescent quencher, dabcyl, or other fluorescent-quenching molecule.

53. An improved assay according to claim 48, wherein the one or more target nucleic acid sequences is 2, each having a distinct SNP of interest.

54. An improved assay according to claim 53, wherein the one or more fourth primers is 2, each having a distinct fluorophore and unique sequence that incorporates into and amplifies each of the 2 target nucleic acid sequences.

55. An improved assay according to claim 48, wherein the one or more target nucleic acid sequences is 3, each having a distinct SNP of interest.

56. An improved assay according to claim 55, wherein the one or more fourth primers is 3, each having a distinct fluorophore and unique sequence that incorporates into and amplifies each of the 3 target nucleic acid sequences.

57. An improved assay according to claim 48, wherein the one or more target nucleic acid sequences is 4, each having a distinct SNP of interest.

58. An improved assay according to claim 57, wherein the one or more fourth primers is 4, each having a distinct fluorophore and unique sequence that incorporates into and amplifies each of the 4 target nucleic acid sequences.

59. A nanoliter sampling array comprising: a) a first platen having at least one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes; wherein each first platen through-hole contains at least i) one or more first primers, each having at least a portion substantially complementary to a first segment of one or more target nucleic acid sequences; and ii) a second primer having at least a portion substantially complementary to a second segment of the one or more target nucleic acid sequences, the first and second primers being ligatable upon binding to the one or more target nucleic acid sequences.

60. A nanoliter sampling array according to claim 59, further comprising: a second platen having at least one hydrophobic surface and having a high- density microfluidic array of hydrophilic second platen through-holes; wherein the first and second platen are fixedly coupled such that the through-holes of each are aligned.

61. A nanoliter sampling array according to claim 59, wherein at least one pair of aligned through-holes contains at least first reagents for a first assay process and second reagents for a second assay process.

62. An array according to claim 61, wherein one of the assay processes is PCR amplification.

63. An array according to claim 61, wherein one of the assay processes is detection of one or more amplified target nucleic acid sequences, each having a SNP.

64. An array according to claim 63, wherein detection of one or more amplified target nucleic acid sequences comprises using one or more probes specific for hybridizing to a region of each of the one or more target sequences, each probe containing a distinct fluorescent group and a fluorescence-modifying group, wherein upon extension of the one or more probes into one or more amplified target nucleic acid sequences, each of the distinct fluorescence-modifying groups is excised and the distinct fluorescent group fluoresces.

65. An array according to claim 63, wherein detection of one or more amplified target nucleic acid sequences comprises using one or more probes specific for hybridizing to a region of each of the one or more target sequences, each probe containing a distinct fluorescent group and a fluorescence-modifying group, wherein the fluorescent group is quenched before incorporation into double-strand product and is dequenched after incorporation into double-stranded product.

66. An array according to claim 65, wherein the fluorescent group is quenched by secondary structure before incorporation into double-stranded product, such that before incorporation, a sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group, quenching the fluorescent group.

67. A nanoliter sampling array according to any of claims 59-66, wherein the primers are affixed on, within or under a coating of the sample through-holes by drying, the coating comprising a biocompatible material.

68. A method of identifying one or more SNPs in one or more target nucleic acid sequences, the method comprising: providing a first sample platen having a high-density microfluidic array of through-holes, each sample platen through-hole containing at least one or more first primers, each first primer having at least a portion substantially complementary to a first segment of the one or more target nucleic acid sequences, a second primer having at least a portion substantially complementary to a second segment of the target sequences, the 5'-end of the second primer ligatable to the 3'-end of the first primer after binding to the one or more target nucleic acid sequences, and a third primer that is substantially complementary to a random sequence segment of the second primer; introducing a sample containing one or more target sequences of nucleic acid, each having a SNP of interest, to the sample platen through-holes in the array; introducing reagents to the sample platen through-holes in the array, the reagents including a reagent for effecting amplification, a reagent for effecting ligation, and at least four different nucleotide bases; effecting ligation of the first and second primers to produce a ligated product; effecting amplification of the ligated product and one or more target sequences; and detecting one or more amplified target sequences.

69. A method of identifying a SNP in a target sequence of nucleic acid according to claim 68, wherein effecting ligation and effecting amplification comprises addition of a ligase and a polymerase followed by subjecting the array to controlled-temperature conditions.

70. A method of identifying one or more SNPs according to claim 68, further comprising, before introducing reagents to the sample platen through-holes in the array: introducing a sample containing one or more probes specific for hybridizing to a region of one or more target nucleic acid sequences and amplifying the one or more target sequences, wherein the one or more probes each contain a distinct fluorescent group and a fluorescence-modifying group.

71. A method of identifying one or more SNPs according to claim 70, wherein upon extension of the one or more probes into one or more amplified target nucleic acid sequences, each of the distinct fluorescence-modifying groups is excised and the distinct fluorescent group fluoresces.

72. An method of identifying one or more SNPs according to claim 70, wherein the fluorescent group is quenched before incorporation into double-strand product and is dequenched after incorporation into double-stranded product.

73. An method for identifying one or more SNPs according to claim 72, wherein the fluorescent group is quenched by secondary structure before incorporation into double- stranded product, such that before incorporation, a sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group, quenching the fluorescent group.

74. A method according to claim 71, wherein identifying one or more SNPs in one or more target nucleic acid sequences comprises momtoring differential fluorescence of the one or more distinct fluorescent groups incorporated into the one or more amplified target nucleic acid sequences.

75. A method of identifying one or more SNPs in one or more target sequences of nucleic acid according to claim 68, wherein the polymerase lacks 5' to 3' exonuclease activity.

76. A method of identifying one or more SNPs in a target sequence of nucleic acid according to claim 68, further comprising using a polymerase that lacks 3' to 5' exonuclease activity.

77. A method of identifying one or more SNPs in one or more target nucleic acid sequences according to claim 68, further comprising using a polymerase that lacks 5' to 3' exonuclease activity and 3' to 5' exonuclease activity.

78. A method according to claim 70, wherein the one or more target nucleic acid sequences is 2, each having a distinct SNP of interest.

79. A method according to claim 78, wherein the one or more hybridizable probes is 2, each having a distinct fluorophore and unique sequence that hybridizes to and amplifies each of the 2 target nucleic acid sequences.

80. A method according to claim 70, wherein the one or more target nucleic acid sequences is 3, each having a distinct SNP of interest.

81. A method according to claim 80, wherein the one or more hybridizable probes is 3, each having a distinct fluorophore and unique sequence that hybridizes to and amplifies each of the 3 target nucleic acid sequences.

82. An improved assay according to claim 70, wherein the one or more target nucleic acid sequences is 4, each having a distinct SNP of interest.

83. An improved assay according to claim 82, wherein the one or more hybridizable probes is 4, each having a distinct fluorophore and unique sequence that hybridizes to and amplifies each of the 4 target nucleic acid sequences.

84. A kit for use in identification of one or more amplified target nucleic acid sequences, the kit comprising: a) a sample platen having one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes; wherein each sample platen through-hole contains at least i) one or more first primers, each first primer having at least a portion substantially complementary to a first segment of one or more target nucleic acid sequences; ii) a second primer having at least a portion substantially complementary to a second segment of the one or more target nucleic acid sequences, the 3 '-end of the one or more first primers ligatable to the 5 '-end of the second primer after binding to the one or more target nucleic acid sequences; b) a reagent platen having a high-density microfluidic array of through-holes, each reagent platen through-hole containing at least i) a third primer that is substantially complementary to a random sequence segment of the second primer; ii) one or more probes specific for hybridizing to a region of one or more target nucleic acid sequences and amplifying the one or more target sequences, wherein the one or more probes each contain a distinct fluorescent group and a fluorescence-modifying group; iii) four different nucleotide bases; iv) a ligase; and the reagent platen having a structural geometry that corresponds to the sample platen allowing delivery of reagent components and target nucleic acid sample to the primers in the sample platen.

85. A kit for use in identification of amplified target nucleic acid sequences according to claim 84, wherein a PCR-compatible buffer is also included.

86. A kit according to claim 84 or 85, wherein the primers are affixed on, within or under a coating of the sample through-holes by drying, the coating comprising a biocompatible material.