WO1992018649A1

WO1992018649A1 - Compositions and methods for improved extraction and hybridization of nucleic acid

Info

Publication number: WO1992018649A1
Application number: PCT/US1992/002976
Authority: WO
Inventors: Jeffrey Van Ness; John C. Tabone; Charles R. Petrie; Trevor H. Adams
Original assignee: Microprobe Corporation
Priority date: 1991-04-12
Filing date: 1992-04-10
Publication date: 1992-10-29

Abstract

Methods for improved RNA/DNA hybridization and for improved extraction of nucleic acid from a biological sample are disclosed. Compositions for improved RNA/DNA hybridization and for improved extraction of nucleic acid are also provided. The hybridization methods make use of a trichloroacetate-containing composition that promotes nucleic acid base pairing and alters the Tm of RNA/DNA duplexes relative to the Tm of corresponding DNA/DNA duplexes. These properties may be advantageously applied in nucleic acid hybridization assays using DNA or RNA oligonucleotide probes. The extraction methods use a trihalogenated acetate-containing composition and permit precipitation of nucleic acid from lysates of complex biological specimens.

Description

COMPOSITIONS ANl5 METHODS FOR IMPROVED EXTRACTION AND HYBRIDIZATION OF NUCLEIC ACID

Technical Field

This invention describes compositions and methods for improved RNA/DNA hybridization and for improved extraction of target nucleic acid from complex biological samples. The compositions contain anionic chaotropes and their respective trihalogenated acetate salts.

Summary of the Invention

A method for improved RNA/DNA hybridization is described. The method includes the steps of combining RNA and complementary DNA with a trichloroacetate-containing composition, thereby forming a hybridization mixture, and incubating the hybridization mixture at a temperature sufficient to optimize the rate of RNA/DNA duplex formation. Compositions for improved RNA/DNA hybridization that include a trichloroacetate salt in a buffer solution having a pH of about 6 to 9 are also disclosed.

Methods and compositions for improved extraction of nucleic acid from a biological sample are also described. These methods include the steps of contacting the biological sample with a trihalogenated acetate- containing composition, thereby releasing nucleic acid in a lysate; combining the lysate with an alcohol in an amount sufficient to precipitate the nucleic acid, thereby forming an aqueous phase having precipitated nucleic acid therein; and removing the aqueous phase from the precipitated nucleic acid. The removing step may be accomplished in the absence of centrifugation through retention of precipitated nucleic acids on a woven fabric. The described extraction method eliminates the disadvantages associated with organic solvents and biphasic separations, and offers the advantages of a simple procedure having a minimal number of manipulative steps.

Detailed Description of the Invention Prior to describing the present invention in greater detail, it may be helpful to define the following terms.

The term "oligonucleotide" refers to a polynucleotide (DNA or RNA) that is preferably 12-100 nucleotides in length, and more preferably 14-50 nucleotides in length.

The term "stringency" refers to a condition for performing nucleic acid hybridization whereby base pair mismatching is controlled (for instance, by temperature, salt concentration, solvent and the like) . Under strictest stringency conditions, base pair mismatching does not occur, and any stable nucleic acid duplex formed is perfectly base- paired.

The term "Tm" refers to the temperature at which 50% of a particular nucleic acid duplex or hybrid is melted (i.e., exists as single strands).

The term "target" refers to a nucleic acid molecule, and includes single stranded or double stranded RNA or DNA. "Target" also includes double strand plasmid DNA, genomic DNA from procaryotic and eucaryotic sources, mRNA, rRNA, viral genomic double strand and single strand RNA and DNA, and the like.

The term "capture oligonucleotide" (or "capture probe") refers to an oligonucleotide that is complementary to a target nucleic acid sequence and that is immobilized on the surface of a solid support. Hybridization with a capture oligonucleotide immobilizes ("captures") target nucleic acid.

The term "solid support" refers to any surface that is transferable from solution to solution and suitable for use in oligonucleotide-based hybridization assays. "Solid support" as used herein includes, but is not limited to, membranes, beads, icrotiter wells, strings, plastic strips or any solid surface onto which oligonucleotides may be immobilized. The term "immobilizing" or "immobilization" refers to binding of a soluble molecule or complex to an insoluble solid support, preferably through interaction of a ligand/receptor combination, more preferably by nucleic acid duplex formation, and most preferably through hybridization with a capture oligonucleotide.

The term "signal oligonucleotide" (or "signal probe") refers to an oligonucleotide that is complementary to a target nucleic acid sequence and has either a detectable label bound thereto or the capability of binding to a moiety with a detectable label bound thereto.

The term "sandwich assay" refers to both solid phase and solution nucleic acid hybridization. For purposes of this description, capture and signal oligonucleotides may be added simultaneously or sequentially. A typical solid phase sandwich assay features target nucleic acid hybridized to both immobilized capture oligonucleotide and signal oligonucleotide, resulting in a "sandwich" of capture probe/target nucleic acid/signal probe.

The term "trihalogenated acetate" refers to a family of chemical compounds that includes trichloroacetic acid, trifluoroacetic acid, tribromoacetic acid and salts thereof.

The term "precipitated nucleic acid" refers to insolubilized nucleic acid, either removed from a liquid or present in a liquid in a spectrum of physical states ranging from (i) insolubilized material invisible to the naked eye to (ii) a relatively coarse dispersion of solid particles to (iii) a solid phase separated from or settled out of solution. Nucleic acid hybridization, a well known technique for detecting and identifying target nucleic acids, features base pairing of complementary nucleic acid strands to form double stranded, stable duplex molecules. The stable duplex molecules formed may be DNA/DNA hybrids, RNA/RNA hybrids or DNA/RNA hybrids.

In general, nucleic acid hybridization involves first combining a single strand target polynucleotide (or a denatured, double strand polynucleotide target) and a complementary single strand polynucleotide probe, and then detecting polynucleotide duplex (target/probe) formation. Conventional hybridization formats include those where the target nucleic acid is initially contained in a cell (in situ. ; those where the polynucleotide target or the oligonucleotide probe is initially immobilized on a solid support (solid-phase hybridization) ; and those where target and probe(s) nucleotide species are in solution (solution hybridization) . For purposes of the present invention, the particular hybridization format is not critical. As improvements in current hybridization formats are made and new formats developed, they can readily be applied to this invention.

In the first aspect of the claimed invention, methods for hybridizing oligonucleotide probes and target nucleic acid using trichloroacetate (TCA)-containing compositions are described. Trichloroacetate promotes nucleic acid base pairing and decreases the Tm of a DNA/DNA oligonucleotide duplex relative to that of the corresponding RNA/DNA oligonucleotide hybrid. Accordingly, if the target nucleic acid is RNA and the oligonucleotide capture and signal probes are DNA, stable RNA/DNA hybrids may be formed at temperatures that melt adventitiously associated DNA/DNA

(capture probe/signal probe) duplexes. This alteration in DNA/DNA Tm (as compared to the DNA/DNA Tm in typical hybridization solutions, such as 0.1S M NaCl and 30% formamide) results in significant reduction of non-specific, background signal produced by capture probe/signal probe association. Empirical observations suggest that approximately 20 to 25% of oligonucleotide capture probe/ signal probe combinations used for the detection of specific target nucleic acid exhibit some degree of non-specific base pairing. For instance, universal signal sequences employed for detection of bacterial 16S rRNA display some amount of non-specific base pairing with 20 to 25% of bacteria- specific oligonucleotides used as capture probes. This non¬ specific base pairing of signal probe/capture probe may lead to unacceptable levels of background in a sandwich assay format.

One factor contributing to the observed non- specific association of oligonucleotide capture and signal probes is the low sequence complexity of the probes (generally about 14 to 50 bases in length) . More typically, the length of an oligonucleotide capture or signal probe is 20 to 24 nucleotides, and by random chance it is expected that, on the average, 7 to 9 base pairs will form between any two probes. Some of these base pair combinations are extremely and unpredictably stable.

A second factor contributing to the observed non-specific association of probes is the high concentration of oligonucleotides employed on both the capture and signal side of the typical sandwich assay format (generally about 0.1 μg to about 1.0 μg capture probe/solid support; about 1 μg/ml signal probe). Since the sensitivity of oligonu¬ cleotide probes is exquisite (capable of detecting less than picogram levels of complementary oligonucleotide) , low levels of non-specifically associated capture/signal probe may lead to very high background signal levels. For example, a non-specific binding frequency of l signal probe per 500,000 capture probes may cause extensive background signal levels. Trichloroacetate (TCA)-containing hybridization solutions of the present invention may also be used for enhanced capture of double strand DNA targets with RNA or "RNA-like" probes. Under standard hybridization conditions (for instance, in 0.18 M NaCl), the Tm of RNA/DNA duplexes is about 15°C below the Tm of corresponding DNA/DNA duplexes, which in turn have a Tm about 15°C below that of corresponding RNA/RNA duplexes. Using standard conditions, if the target nucleic acid is double stranded DNA and the capture and/or signal probes are RNA (or possess "RNA-like" properties [e.g., 2*-O-methyloligoribonucleotides, as described by Inoue et al., Nucl. Acids Res. 15;6131-48. 1987]), renaturation of target nucleic acid (DNA/DNA pairing) is favored over probe/target (RNA/DNA) hybridi- zation. Thus, under standard hybridization conditions, target renaturation competes with probe hybridization. This target renaturation phenomenon becomes more problematic when the target nucleic acid is of relatively low sequence complexity (for instance, less than 20 kilobases in length) , because the rate of renaturation is directly proportional to sequence complexity.

The following illustration may be instructive.

A target nucleic acid sample contains 10⁹ copies of an 11 kilobase (Kb) plasmid DNA in a 100 μl volume. The molecular weight of an 11 Kb plasmid is about 7 x 10^e daltons (using a calculation of 660 daltons per base pair) . Further assuming approximately l x 10¹⁵ base pairs'per μg of DNA, the target nucleic acid in the sample represents about 0.011 μg of

DNA/100 μl, or about 0.11 μg DNA/ml. This amount of target nucleic acid is equivalent to about 0.0024 A₂₆₀ optical density units (conversion = 1 A₂₆₀ unit per 45 μg DNA/ml) .

The rate of renaturation of the double strand plasmid is represented by the Cot value [Cot -= A₂₆₀/2 x time (in hours)], therefore the plasmid would renature at a rate of about 0.0012 Cot units per hour under stringent conditions. Since long nucleic acid fragments renature more quickly than short fragments, the calculated Cot value is corrected for nucleic acid fragment length. The rate of renaturation is proportional to the square root of the ratio of fragment length to a reference length. The reference length is 400 bp, since Cot_1/2 is compared to the renatur¬ ation of E__, coli DNA that has been sheared to fragments approximately 400 nucleotides in length. Within this illustrative example, the correction factor is 5.2 (square root of 11,000/400), therefore renaturation of the plasmid is occurring at 0.0062 Cots/h. J . coli genomic DNA has a Cot_1/2 = 3.2, with a complexity of 4,200,000 bp. The Cot_1/2 of the plasmid is calculated as 3.2/4,200,000 = x/11,000, where x - 0.00838. The plasmid will therefore reach Cot_1/2 in about 1.3 h (the time at which it is 50% renatured) , if the reaction is performed at about 15°C to about 25 °C below Tm for DNA/DNA.

These calculations represent the potential renaturation of an 11 Kb double strand DNA target during a standard hybridization assay.

Accordingly, suppression of DNA/DNA renaturation through use of a TCA-containing hybridization solution may be advantageous, especially when the double strand target DNA is present in relatively high concen¬ trations. That is, the presence of TCA significantly decreases DNA/DNA renaturation that competes with target/ probe hybridization. Further, a depression of DNA/DNA Tm may be desirable when capturing a single strand of denatured target DNA with either an immobilized RNA probe or an "RNA- like" probe.

Within this description, exemplary "RNA-like" oligonucleotide probes include 2•-O-methyloligoribo- nucleotides, as described by Inoue et al., Nucl. Acids Res. 2_5i6131-48, 1987. A 2'-O-methyloligoribonucleotide is an RNA analog that has a methylated 2'-hydroxyl at the ribose sugar. A 2•-O-methyloligoribonucleotide (2*0-Me) capture probe may be used for hybridizing with a DNA target, and the Tm for the resulting 2'0-Me capture probe/DNA target duplex is about 15^βC to about 20°C above that of the corresponding DNA/DNA duplex. Further, 2-O-Me probes exhibit thermal stability characteristics equivalent to RNA probes in forming RNA/RNA duplexes. Thus, the use of 2'0-Me analogs in synthesis of oligonucleotide probes results in an alteration of relative Tm's — 2'0-Me/RNA > 2'O-Me/DNA > DNA/DNA — similar to that observed with hybridization in TCA-containing solutions.

The 2'0-Me probes may be used in combination with a TCA hybridization solution to facilitate capture or signal probe hybridization with a denatured double strand DNA target. More specifically, prevention of self rena¬ turation of a double strand DNA target permits capture of single strand target DNA by 2-O-Me probes at or near the melting temperature of the target nucleic acid. Additional advantages provided by 2-O-Me probes include resistance to nucleases (RNAses and DNAses) and alkali stability (B.S. Sproat et al., Nucl. Acids Res. 17.3373-86. 1989).

Within the present invention, preferred trichloroacetate-containing compositions include rubidium, sodium, potassium, cesium and lithium trichloroacetate (RbTCA, NaTCA, KTCA, CsTCA, LiTCA) , preferably used at a concentration of about 2 molar to about 5 molar. Any chaotrope capable of decreasing the Tm of a DNA/DNA duplex about 40^βC to about 50°C below the Tm of the same DNA/DNA duplex in 0.18 M NaCl may also be suitable for use within the claimed invention. TCA-containing compositions decrease the Tm of DNA/DNA duplexes below the Tm of the corresponding RNA/DNA hybrids in the same TCA-containing composition, making solutions of trichloroacetate particularly useful for DNA probe/target RNA/DNA probe sandwich hybridization assays. One of ordinary skill in the art of nucleic acid hybridization will appreciate that specific nucleotide sequence effects, GC ratio and nearest neighbor phenomena may affect the Tm calculated for a particular sequence and its complement. These characteristics, as well as other predetermined criteria (such as stringency, sequence length and the like) , are considered in selecting a temperature of hybridization. For instance, the selected temperature for RNA/DNA hybridization in a TCA-containing solution may vary between about -10 °C to about -l^βC from the Tm of the desired RNA/DNA duplex in that TCA-containing solution.

If the target nucleic acid is RNA (rRNA, single strand RNA, or double strand RNA) and the hybridization solution contains TCA, stringency conditions may be selected to permit specific capture of RNA target by DNA capture oligonucleotides at or above the melting temperature of DNA/DNA duplexes. In this case, any background signal arising from non-specific association of DNA signal and DNA capture oligonucleotides is greatly reduced. If the target nucleic acid is double strand DNA and the hybridization solution contains TCA, stringency conditions can be selected so as to permit hybridization of RNA probe and DNA target at or near the melting temperature of the double strand DNA target. Since a TCA-containing composition in combination with appropriate temperature selection may prevent self renaturation of target DNA, target renaturation that competes with RNA probe(s)/DNA target hybridization may be minimized or avoided. TCA-containing compositions also permit low temperature denaturation (about 35°C to about 60°C) of double strand RNA or DNA target nucleic acid. A second aspect of the claimed invention discloses improved methods for extraction of nucleic acids from a complex biological sample. Compositions containing a trihalogenated acetate (THA) release target nucleic acids from cells present in a biological sample, thereby forming a THA lysate. Preferred trihalogenated acetate compositions suitable for use within the present invention include lithium trichloroacetate, potassium trichloroacetate, sodium trichloroacetate, lithium tribromoacetate, potassium tribromoacetate and sodium tribromoacetate. Addition of an alcohol or a mixture of alcohols to the THA lysate results in an aqueous phase that contains precipitated nucleic acid. Exemplary alcohols are water-miscible, with ethanol, isopro- panol or mixtures thereof preferred. The aqueous phase is then removed from the precipitated nucleic acid (for instance, by centrifugation) .

The present invention also describes an alter¬ native method for isolating and purifying precipitated nucleic acids from solution without the need to perform a centrifugation step. This alternative method involves wicking the aqueous phase containing precipitated nucleic acids through a woven fabric. The precipitated nucleic acids are retained on the fabric (preferably a piece of fabric of predetermined size) , and may be recovered by placing the fabric piece with retained nucleic acids in an aliquot of distilled water. Yields of nucleic acids recovered by this alternate method are comparable to yields recovered by centrifugation of precipitated nucleic acids.

The described THA extraction methods and compositions are compatible with isolation of low copy number target nucleic acid from biological specimens prior to nucleic acid amplification. As compared to typical phenol-chloroform extraction procedures, the claimed methods and compositions are environmentally safe. The THA extraction methods described herein require fewer manipu¬ lative steps than typical phenol-chloroform extraction procedures, and thus the claimed extraction methods can be performed relatively rapidly (i.e., "hands-on" time is reduced) . In addition, improved yields of nucleic acids may be obtained, since nucleic acid exposure to nucleases released into target cell lysates is minimized by the short extraction time. Moreover, THA solutions can inactivate infectious particles (such as viruses) , so extraction of infectious biological specimens by laboratory personnel is made safer. In practice of the claimed THA extraction methods, phase separation is not required for isolation of nucleic acids (in contrast to a typical phenol-chloroform nucleic acid extraction procedure) . Through elimination of phase separation, transfer of liquid phase(s) from vessel to vessel is avoided. As a result, the following advantages are obtained: (1) the amount of liquid handling during the extraction procedure is significantly reduced; (2) the imprecision of liquid-interphase separation and liquid phase transfer is avoided; (3) the loss of low quantities of nucleic acids at the interphase is eliminated; and (4) the recovery of nucleic acids is improved. The claimed extraction methods provide further advantages, such as easy adaptability to automation and savings of time, labor and equipment costs. Either the THA extraction methods or the woven fabric separation of precipitated nucleic acids (or both) may be advantageously combined with nucleic acid amplification. Elimination of phase separation according to the extraction methods described herein decreases the possibility of contaminating isolated nucleic acids with organic solvent. This type of contamination is a significant problem with nucleic acid preparations that are subjected to amplification procedures, such as polymerase chain reaction (PCR) . Further, the fabric piece with retained, precipitated nucleic acids can be placed directly into an amplification solution or mixture, thereby minimizing loss of low quantities of isolated nucleic acids. Many woven fabrics are suitable for use within the present invention. Preferred woven fabrics have a homogeneous fiber diameter and can be precision woven. Fabrics that are minimally "sticky" and possess neutral chemical properties are also preferred. Particularly preferred woven fabrics include nylon or polyester fabric having a mesh range of about 1 to about 100 microns. One of ordinary skill in the art of nucleic acid isolation, hybridization and/or amplification will appreciate that other woven fabrics may be useful in practice of the present invention, and that many other uses of the woven fabric are contemplated (for instance, precipitated protein or antigen- antibody complexes may be retained on a woven fabric piece) .

THA-containing compositions are used to extract total nucleic acid (DNA and RNA) from biological samples. Preferably, standard buffers and/or detergents are included in THA extraction solutions to promote release of target nucleic acids from cells. Exemplary buffers include sodium citrate, Tris-HCl, PIPES and HEPES, with Tris-HCl at a concentration of about 0.05 M to about 0.1 M preferred. An exemplary THA extraction solution will typically include about 0.05% to about 0.5% (w/v) nonionic or ionic detergent, such as N-lauroylsarcosine, sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate (Sigma Chemical Co., St. Louis, MO), and may further include a chelate at a concentration from about 1 to about 10 mM. - Preferred chelates include sodium ethylenediaminetetraacetic acid (EDTA) , lithium EDTA, sodium ethylene glycol-bis(fl-aminoethyl ether) N,N,N',N*-tetra- acetic acid (E6TA) and lithium E6TA.

The extraction methods and compositions described herein may be advantageously used prior to performing hybridization with extracted target nucleic acid, and especially hybridization with nucleic acid obtained from a complex biological sample, such as feces or blood) . For some samples, the claimed extraction procedure may be advan¬ tageously used to remove contaminants that contribute to background interference in hybridization assays. In addition, the THA extraction method used in combination with procedures that concentrate target nucleic acid- may improve sensitivity and signal-to-noise ratio in hybridization assays.

The extraction and hybridization methods of the present invention may be applied to complex biological mixtures containing both nucleic acid (RNA and/or DNA) and non-nucleic acid components. Exemplary complex biological mixtures include a wide range of eucaryotic and procaryotic cells, including protoplasts, as well as other cultured or natural biological materials. For instance, the claimed methods may be advantageously used with cultured animal cells, animal tissue (e.g., heart, liver or brain tissue homogenized in lysis buffer), blood cells, retiσulocytes, lymphocytes, plant cells and other cells sensitive to osmotic shock, as well as bacteria, yeast, viruses, mycoplasmas, protozoa, rickettsia, fungi, other small microbial cells and the like. Biological samples of interest include virtually any type of biological specimen that may contain one or more pathogens of interest. In addition, the methods of the claimed invention are useful for detecting non-pathogenic microorganisms, cells, plasmids, viruses, phages or nucleic acid sequences in a specimen. Using the claimed methods for extracting nucleic acid in combination with methods for hybridizing specific oligonucleotide probes and nucleic acids resident in a biological sample, target cells, organisms, viruses or nucleic acid sequences within the sample may be detected.

The hybridization methods of the present invention may also be used to detect or quantitate nucleic acid that is lysed, isolated and/or purified by other means. The lysate or nucleic acid sample subjected to hybridization may include varying amounts of non-nucleic acid components (for instance, from about 0% to about 99% non-nucleic acid) .

Within the present invention, the hybridization format employed is not critical. Therefore, as improvements in hybridization formats and new formats are developed, they can readily be used within the present invention.

In instances where the hybridization format involves a solid support, the solid support may consist of a membrane-type solid support (two dimensional solid support) , a bead-type solid support (three dimensional solid support) or other solid surface, such as a microtiter well.

Various labels (signals) are suitable for use within the present invention. Labels act as reporter groups for detecting duplex formation between a target sequence and its complementary signal sequence. A reporter group as used herein is a group which has a physical or chemical charac¬ teristic which can be measured or detected. Detectability may be provided by such characteristics as color change, luminescence, fluorescence, or radioactivity; or it may be provided by the ability of the reporter group to serve as a ligand recognition site.

Oligonucleotide probes may be labeled by any one of several methods typically used in the art. A common method of detection uses autoradiography and ³H, ¹²⁵I, ³⁵S, "C or ³²P labeled probes or the like. Other reporter groups include ligands (for instance, antigens) which bind to receptors (for instance, antibodies) labeled with fluoro- phores, chemiluminescent agents, enzymes or the like. Alternatively, oligonucleotide probes can be conjugated directly with labels such as f uorophores, chemiluminescent agents, enzymes and enzyme substrates. The choice of label depends on sensitivity required, ease of conjugation with the probe, stability requirements, available instrumentation and the like. Non-isotopic probes may be labeled directly with signal (e.g., fluorophore, chemiluminescent agent, enzyme or the like) , or labeled indirectly by conjugation with a ligand capable of binding a moiety having a detec- table signal bound thereto. For example, biotiη covalently bound to a probe can bind streptavidin that is covalently bound to a detectable signal, such as an enzyme or a photo- reactive compound. Ligand and receptor combinations may be varied widely. Where a ligand has a natural "receptor" (i.e., ligands such as biotin, thyroxine, and cortisol) , it may be used in conjunction with its labeled, naturally occurring receptor. Alternatively, a hapten or antigen may be used in combination with a suitably labeled antibody.

Enzymes suitable for use as signals include hydrolases (particularly phosphatases) , esterases, ureases, glycosidases, and oxidoreductases (particularly peroxi- dases) . Suitable fluorescent signals include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone and the like. Chemiluminescers useful within the claimed invention include luciferin, luminol and 1,2 dioxetanes.

The amount of labeled probe which is present in the hybridization solution may vary widely. Generally, substantial molar excesses of probe over the amount of target nucleic acid will be employed in order to enhance the rate of binding of the probe to the target nucleic acid.

The means of -detecting signal is determined by the signal selected. For example, when the label is a radioisotope, the support surface containing captured and labeled duplexes may be exposed to X-ray film or analyzed in a scintillation or gamma counter. When the label is fluorescent, duplexes are irradiated with light of a particular wavelength that is absorbed by the labeled duplex, resulting in emission of light of a different wavelength which is detected. When the label is an enzyme. duplexes are incubated with an appropriate substrate for the enzyme, and the signal generated may be, for example, a colored precipitate, a colored or fluorescent soluble material, or photons generated by bioluminescence or chemiluminescence.

Detection of duplexes may require binding of a signal generating complex to duplexes of target and oligonucleotide probe(s) . Typically, this binding occurs through ligand-receptor interactions, such as interaction of a ligand-probe conjugate and a receptor-signal conjugate.

The selected label may also allow indirect detection of duplex formation. For example, where the oligonucleotide probe is conjugated with a hapten or antigen, duplexes can be detected by using antibody-signal conjugates. The signal conjugates may include fluorescent molecules, enzymes or radioisotopes.

The following examples are offered by way of illustration and are not to be construed as limiting the invention, as claimed, in any way.

EXAMPLE 1 General Methods and Materials

Materials

FW (filter wash) is 0.09 M NaCl, 50 mM Tris (pH 7.6), and 25 mM EDTA.

SDS/FW is FW and 0.1% (w/v) sodium dodecyl sulfate (SDS) . HEP (horseradish peroxidase) substrate solution is 0.5 mg/ml 4-methoxy-l-naphthol (4-MN) , 0.02 mg/ml 3-methyl-2-benzothiazolinone hydrazone and 0.014% (v/v) hydrogen peroxide in 0.1 M sodium citrate (pH 6.5) and 0.2 M sodium phosphate. GuSCN lysis and hybridization solution is 3 M guanidinium thiocyanate, 2% (w/v) N-lauroylsarcosine (sarcosyl) , 50 mM Tris (pH 7.6), and 25 mM EDTA.

CAP buffer is 0.1 M sodium citrate and 0.2 M sodium phosphate (pH 6.5).

Poly(ethyleneimine) was purchased from Polysciences (Warrington, PA) . Burnished or unpolished nylon beads were purchased from Precision Ball Company (Chicago, IL) and The Hoover Group (Sault St. Marie, MI) . Triethyloxonium tetrafluoroborate, hexanediamine, phenylenediamine, succinic anhydride and N-methyl- pyrrolidinone (N-methyl-pyrrolidone, m-pyrol) , were purchased from Aldrich Chemical (Milwaukee, WI) . Polyeth- oxyethanol sorbitan monolaureate (Tween 20®) was purchased from Pierce (Rockford, IL) . Guanidinium isothiocyanate (GuSCN) was purchased from Kodak (Rochester, NY) . The di- and triamines EDR-148, ED-400, ED-6000, and T-3000 were a gift from Texaco Chemical Company (Houston, TX) .

Oliσodeoxynucleotide sequences

["X" indicates a 5'-tethered hexyl amine.] BgO06: 5'-XCCTTAGGACAGTCTTCCTTCACGC-3• BgOOl: 5^»-XCAATACTCGTATCGCCCGTTATTC-3* (Ba-specific capture sequence)

Bg501 5 « -XGAATAACGGGCGATACGAGTATTG-3 •

UP041: 5 ' -XCTGCTGCCTCCCGTAGGAGT- 3 • ( univers a l bacterial signal sequence) UP541: 5 ' -XACTCCTACGGGAGGCAGCAG-3 ^• (complement of UP041)

UP007: 5--XGTATTACCGCGGCTGCTG-3' (universal bacterial signal sequence) UP003A: 5•-TGACGGGCGGTGTGTACAA-3' (universal bacterial signal sequence) Methods

Oligonucleotide synthesis:

Oligodeoxynucleotides complementary to conserved or hypervariable regions of 16S ribosomal RNA (rRNA) of either Actinobacillus actinomycetemcomitans (Aa) , Bacteroides αinαivalis (Bg; also known as Porohyromonas qjn ivalis [Pg])_. Bacteroides intermedius (Bi) , Eikenella corrodens (Ek) , Fusobacterium nucleatum (Fn) , or Wolinella recta (Wr) were synthesized using phosphoramidite chemistry on either an ABI 380B or a Milligen 7500 automated DNA synthesizer. The oligonucleotides were prepared using the standard phosphoramidite chemistry supplied by the vendor and were purified by adaptations of standard methods. Oligonucleotides with 5'-trityl groups were chromatographed on HPLC using a 12 μm, 300 A Rainin (Woburn, MA) Dynamax C-8 4.2 x 250 mm reverse phase column using a gradient of 15% to 55% (v/v) acetronitrile in 0.1 N triethylammonium acetate (pH 7.0), over 20 min. When detritylation was performed, the oligonucleotides were further purified by gel exclusion chromatography. Analytical checks for the quality of the oligonucleotides were conducted with a Toso-Haas DEAE-NPR column at alkaline pH and by polyacrylamide gel electro¬ phoresis (PAGE) .

Preparation of a pol mer-coated n lon bead

25,000 3/32-inch diameter unpolished nylon beads were placed in a flask containing 1800 ml of 100% anhydrous N-methyl-pyrrolidinone and mixed for 5 min at ambient temperature. 200 ml of l M triethyloxonium tetra- fluoroborate in dichloromethane were added and the mixture was stirred for 30 min at ambient temperature. The beads were then decanted and washed quickly with 4 x 500 ml changes of 100% N-methyl-pyrrolidinone. The beads were transferred to 1 1 of 3% (w/v) 10,000 MW poly(ethyleneimine) (prepared from a 30% aqueous solution of poly(ethylenei ine) in N-methyl-pyrrolidinone) , and were tirred for 12 to 24 h at ambient temperature. The beads were washed sequentially with 2 1 N-methyl-pyrrolidinone, 1 1 SDS/FW, and 10 x 2 1 distilled water. The beads were then dried under high vacuum for 4 to 5 h without the use of heat.- The amine content of the beads was determined by reaction with picrylsulfonic acid.

Preparation of 4.6-dichlorotriazine-derived oligonucleotides Ten to 1000 μg of 5•-aminohexyl oligonucleotide were reacted with an excess of recrystallized 2,4,6- trichlorotriazine in 10% N-methyl-pyrrolidinone or 10% acetonitrile in sodium borate buffer (pH 8.3 to 8.5) at about 19°C to about 25^βC for about 30 to about 120 min. Preferably, the final reaction conditions were 0.5 M sodium borate (pH 8.3), 2 mg/ml recrystallized 2,4,6-trichloro- triazine and 500 Mg/ml aminohexyl-oligonucleotide.

Unreacted 2,4,6-trichlorotriazine was removed by size exclusion chromatography on cross-linked dextran (i.e., Sephadex® G-50, Pharmacia, Uppsala, Sweden) . The reaction product was 4,6-dichlorotriazine-derived oligonucleotide.

Preparation of oliαonucleotide-derived nylon beads

Poly(ethyleneimine)-coated nylon beads (described above) were placed in an equal volume of 0.1 M sodium borate (pH 8.3) at 4^βC and vigorously agitated for 30 min to rehydrate the beads. Purified 4,6-dichlorotriazine- derived oligonucleotide was then added to the poly- (ethyleneimine)-coated beads, and the mixture was vigorously agitated at ambient temperature (about 19^βC to about 23°C) for 60 min. The beads were then washed twice with 0.1 M sodium borate (pH 8.3). Succinic anhydride was added at a concentration of 10 mg/ml in 90% N-methyl-pyrrolidinone and 0.1 M sodium borate (pH 8.3), using a volume three times that of the volume of the beads. The reaction was allowed to proceed for 1 h at ambient temperature. The beads were then washed 3 times with 250 ml of 100% N-methyl-pyrro¬ lidinone, twice with distilled water, 5 times with 250 ml SDS/FW, and then 4 times with 1 1 of distilled water. Beads were stored in 25 mM EDTA.

Lysis of bacteria and hybridization conditions

1 x 10⁸ cells of Bacteroides qinoivalis (Bg) were lysed in 100 μl of GuSCN lysis and hybridization solution at 19°C. The cell lysate was heated in a 65°C water bath for 10 min. Biotinylated probe was added to the lysate solution and to the diluent (GuSCN lysis and hybridi¬ zation solution) to a final concentration of 100 ng/ml, and 5 to 8 5-fold serial dilutions were made of the starting lysate. The solutions were incubated with the oligonucleotide-derived nylon bead 1 h at ambient temperature with mild agitation. The solid supports were then washed once with the GuSCN lysis and hybridization solution, once with FW, and once with SDS/FW. Streptavidin/HRP conjugate was added to a final concentration of 1 μg/ml (based on streptavidin) in SDS/FW and incubated 10 to 15 min at ambient temperature with mild agitation. The beads were then washed three times with SDS/FW and once with CAP buffer. HRP substrate solution was added, and the reaction was allowed to proceed for 15 min at ambient temperature. The beads were then washed once with SDS/FW, once with FW, and allowed to air dry in the dark.

Quantitative determination of the extent of hybridization (capture of tarσet nucleic acid^ using insoluble substrates for either horseradish peroxidase or alkaline phosohatase

After completion of the sandwich hybridization assay and deposition of an insoluble colored product (resulting from the reaction of HRP and the HRP substrate solution) onto the surface of the beads, as described above, the quantity of deposited product was determined by fluore¬ scence quenching. The beads were dried for 15 to 30 min at ambient temperature and then placed in a round bottom opaque microtiter plate (Dynatek Laboratories, Chantilly, VA) . The beads were read using a fluorometer (Fluoroskan II, Flow Laboratories, McLean, VA) , in which excitation was at 350 nm and emission was at 456 nm. Fluorescence quenching tech¬ niques have been described in copending patent application USSN 07/558,967.

Determination of Tm values in various hybridization solutions or chaotropic salts

For the determination of DNA/DNA Tm's, ³²P- labeled UP041 oligonucleotide (3'-labeled with terminal transferase) was incubated with a complementary DNA oligonucleotide (UP541) immobilized on nylon beads. Five to 50 ng of ³²P-UP041 were hybridized in 300 μl volumes of various hybridization solutions at ambient temperature for 30 min. The beads were then washed three times with 1 ml of the respective hybridization solution, 3 times with SDS/FW, and then once more with the respective hybridization solution. The beads were placed in a 15°C water bath in 300 μl of respective hybridization solution. At 5 min intervals, the temperature was raised 5^βC, the solution decanted into a scintillation vial, and fresh respective hybridization solution added to the beads. The respective hybridization solutions were raised in temperature concurrently with the beads. The Tm melt occurred over approximately a 15^βC to 80°C temperature range. Radioactivity was determined by scintillation counting.

To calculate the Tm, cumulative counts eluted at each temperature were plotted against temperature, and the temperature at which 50% of the material was eluted from the bead was taken as the Tm.

For the determination of RNA/DNA Tm's, rRNA was captured on Bg016-derived nylon beads in the presence of ³²P-labeled signal oligonucleotide (UP041) . The beads were then washed and processed as described above. EXAMPLE 2

Rubidium Trichloroacetate-basβd Hybridization Solutions and Promotion of Specific Nucleic Acid Base Pairing

A solution containing 3 M rubidium trichloro¬ acetate (RbTCA), 50 mM Tris (pH 7.6), 25 mM EDTA and 2% (w/v) N-lauroylsarcQsine was used to determine the specificity of hybridization of ³²P-labeled specific oligonucleotide probes (i.e., synthetic oligonucleotide probes having sequences complementary to unique hyper- variable regions of 16S rRNA of various bacteria) with total nucleic acid from Aa, Bg, Bi, Ek, Fn, Wr and E^. coli immobilized on nylon membrane, as described above. Approxi¬ mately 50 ng of total nucleic acid were immobilized per each nylon membrane slot. A ³²P-labeled probe complementary to Aa (6.5 x 10⁶ cpm; approximately 65 ng) was added to 1 ml of the RbTCA solution described above, and incubated with a membrane slot containing either Aa, Bg, Bi, Ek, Fn, Wr, or E. coli nucleic acid. Hybridization was allowed to proceed for 3 h at 19^βC. The filters were washed with 0.09 M NaCl, 50 mM Tris (pH 7.6), 25 mM EDTA and 0.1% (w/v) SDS at 60°C, and radioactivity . was detected by autoradiography. The Aa-specific probe hybridized exclusively with membrane slots immobilizing Aa total nucleic acid, demonstrating that the 3 M RbTCA solution promoted specific nucleic acid base pairing.

■EXAMPLE 3 Specific Detection of Bacteroides qingivalis in a RbTCA-based Hybridization Solution

A 100 μl volume of RbTCA lysis/hybridization solution (3 M RbTCA, 50 mM Tris (pH 7.6), 25 mM EDTA, and 2%

(w/v) N-lauroylsarcosine) was used to lyse a pellet consisting of 1 x 10⁸ cells each of Aa, Bg, Bi, Ek, Fn, or

Wr at 19^βC. The lysate was diluted from 1 x 10⁸ to 6.4 x 10³ Bg cell equivalents in 5-fold increments using a diluent containing (i) a biotinylated 24-mer oligonucleotide probe complementary to conserved regions of bacterial 16S rRNA (signal probe) at a final concentration of 100 ng/ml and 5 (ii) a mixture of 1 x 10⁸ lysed cells of each of Aa, Bi, Ek, Rn and Wr. Each mixture was incubated for 1 h at ambient temperature with a Bg-specific oligonucleotide probe (capture probe; 0.2 μg) covalently immobilized on 3/32-inch diameter nylon beads. The solid supports were washed with

10 SDS/FW at ambient temperature and incubated with 10 ng/ml of streptavidin/horseradish peroxidase (SA/HRP) conjugate in SDS/FW for 30 min at ambient temperature. The solid supports were then washed with SDS/FW and FW, and the presence of HRP was determined by incubating the supports

15 with HRP substrate solution. The beads were washed once with water, dried in the dark, and fluorescence per bead was determined (as described in Example l) . Only Bg target nucleic acid was detected in the colorimetric sandwich assay. Thus, the RbTCA hybridization solution promoted

20 effective lysis and specific nucleic acid base pairing of the capture and signal probes with the target nucleic acid.

TABLE 1

Specific Detection e v ^•25 in a RbTCA-basi

Cell Number

5 x 10⁷

1 x 10⁷ 30 2 X 10⁶

4 x 10⁵

8 X 10*

1.4 X 10*

3.2 X 10³

35 none

^* = lower level of detection as determined by the 3 x SD test. EXAMPLE 4

Suppression of DNA/DNA T in

TCA-containing Solutions

Table 2 compares the Tm of nucleic acid duplexes in RbTCA and other hybridization solutions.

TABLE 2 Melting Temperatures

19-mer probes were used for DNA/DNA hybridizations; 24-mer probes were used for RNA/DNA hybridizations; RNA is 16S rRNA.

Tm values were determined as described in Example 1.

As shown in Table 2, the Tm of the DNA/DNA hybrid in RbTCA is 43°C below the Tm of the same hybrid in 0.18 M NaCl, and 27°C below the Tm of the same hybrid in 30% formamide. Further, the DNA/DNA Tm in RbTCA is 14°C below that of the same hybrid in 3 M GuSCN, while the Tm of the corresponding RNA/DNA hybrid is the same in either GuSCN or RbTCA. The difference between the Tm's for DNA/DNA and RNA/DNA hybrids in 3 M GuSCN is not significant enough to permit preferential formation of RNA/DNA hybrids. However, these data indicate that the Tm of DNA/DNA duplexes is significantly suppressed as compared to RNA/DNA hybrids in RbTCA-containing solutions. This difference in Tm's is large enough to permit preferential formation of RNA/DNA hybrids. Thus, double strand DNA targets may be captured with an RNA or "RNA-like" probe under conditions where competing DNA/DNA renaturation reactions are greatly slowed with respect to the rate at which RNA/DNA hybrids form.

EXAMPLE 5

Reduction of Signal Oligonucleotide-associated Background by Performing Hybridization Assays in TCA-based Solutions

UP003 and BgOOl probes form non-specific base- paired duplexes when BgOOl is immobilized on solid supports as a capture oligonucleotide and UP003 is biotinylated and used as a signal oligonucleotide in a sandwich assay format. The BgOOl sequence is 5'-XCAATACTCGTATCGCCCGTTATTC-3• , and the UP003 sequence is 5•-TGACGGGCGGTGTGTACAA-3• . These sequences can associate to form the following non- specifically base paired duplex:

BIOTIN - 5'-TGACGGGCGGTGTGTACAA-3' (UP003 sequence) * ******* * * * 3*-CTTATTGCCCGCTATGCTCATAAC-5' (BgOOl sequence)

where 11 of the 19 possible base pairs are properly formed. While 11/19 base pairs would not be expected to yield a stable duplex under stringent hybridization solutions (for instance, a 3 M GuSCN hybridization solution) , Table 3 demonstrates that stable base pairing does occur in 3 M GuSCN. This "non-specific" hybridization can be eliminated or greatly reduced through the use of a 3 M TCA-containing hybridization solution. TABLE 3 Signal Strength in 3 M GuSCN vs. 3 M RbTCA

M GuSCN H br d a S

Table 3 compares the extent of duplex formation in 3 M GuSCN and 3 M RbTCA. The duplexes represent a perfectly base paired set of sequences (biotin-Bg00l/Bg501; positive control) , the biotin-UPO03/BgOOl (11/19 base pair) combination described above, and a biotin-UP041/Bg001 combination (negative control) .

Briefly, biotinylated oligonucleotides (Bg501, UP003 or UP041) were added to 500 μl of either 3 M GuSCN or 3 M RbTCA hybridization solution to a final concentration of 1 μg/ml. Three BgOOl oligonucleotide-derived beads were added to each tube and incubated at 2l^βC for 30 min. The solid supports were washed once with the respective hybridization solution, once with FW, and once with SDS/FW. Streptavidin/ HRP conjugate was added to a final concen¬ tration of 1 μg/ml (based on streptavidin) in SDS/FW and incubated 10 to 15 min at ambient temperature with mild agitation. The beads were then washed three times with SDS/FW and once with CAP buffer. HRP substrate solution was added, and the reaction was allowed to proceed for 15 min at ambient temperature. The beads were then washed once with SDS/FW, once with FW and allowed to air dry in the dark. The extent of 4-MN product deposited on the bead was measured by the fluorescence quenching technique described in Example 1. Table 3 shows that the background produced by non-specifically hybridized Bg001/UP003 probes in a 3 M GuSCN hybridization solution was completely eliminated with the 3 M RbTCA hybridization solution.

EXAMPLE 6

Lysis of Bacterial and Human Cells with a TCA-based Solution

A pre-prepared TCA lysis solution (250-500 μl lysis solution per 50 μl sample) containing 3 M LiTCA or RbTCA, 50 mM Tris (pH 7.6), 25 mM EDTA, and 2% (w/v) N- lauroylsarcosine was used to lyse 1 x 10⁹ cells of Actinobacillus actinomvcetemcomitans ("Aa") , Bacteroides intermedius ("Bi") , Eikenella corrodens ("Ek") , Wollinella recta ("Wr") , Fusobacterium nucleatum ("Fn") , Bacteroides ginqivalis ("Bg") , or 1 x 10⁶ HeLa cells in 100 μl volumes each. The lysis mixture cleared within 10 seconds, and phase contrast microscopy indicated solubilization of all of the cell types tested. Further, no intact cell shapes were observed by light microscopy. In addition, the lysates were examined by fluorescence microscopy, wherein ethidiu bromide was added to the lysate at a concentration of 0.5 μg/ml. Only extremely diffuse staining was observed in all lysates compared to intact cell controls, indicating that lysis was complete and that chromosomal DNA was unfolded and released from the cells. EXAMPLE 7

Puri ication of Total Nucleic Acid from Bacterial Cells

Solubilized with 3 M LiTCA

To recover total nucleic acid • from LiTCA- solubilized bacterial cells, approximately 1 x 10⁹ to 1 x 10⁷ Bg cells were lysed in 400 μl of 3 M LiTCA, 50 mM Tris (pH 7.6), 25 mM EDTA and 1% (w/v) LiDS. Ethyl alcohol or a benzyl alcohol/ ethanol mixture (800 μl) was added to the lysate and the mixture was mixed well. The mixture was then chilled at -20°C for 20 min. Nucleic acid was pelleted by centrifuging for 5 min at 20,000 x g at ambient temperature, and the resulting pellet was solubilized with 100 μl of water. An aliquot (30 μl) of the nucleic acid solution was examined by gel electrophoresis. The results (Figure 1) indicate that chromosomal DNA and rRNA were efficiently extracted from the LiTCA-solubilized bacterial cells. When compared to the traditional phenol extraction procedure, LiTCA extraction provides better yields of nucleic acid from low numbers of bacteria.

EXAMPLE 8 LiTCA Isolation of Low Copy Number DNA from Blood and Urine Prior to Amplification

A. Urine

Multiple 1 ml aliquots of fresh urine in 1.5 ml microcentrifuge tubes were spiked with 20 μl of growth medium containing 0, 2 x 10², 2 x 10³, or 2 x 10* H, pylori . These spiked aliquots were then centrifuged 3 min in a microcentrifuge to pellet the bacteria, and the resultant pellets were used as a concentration panel to evaluate the LiTCA extraction protocol. The LiTCA protocol involved dissolving each bacterial pellet in 100 μl of water containing 4 μg of homochromatography mix (partially hydrolyzed tRNA used as a carrier) , adding 250 μl of LiTCA lysis solution (3 M LiTCA, 50 mM Tris (pH 7.6), 10 mM LiEDTA, and 2% (w/v) N-lauroylsarcosine) , and vortexing. To the lysate was added 700 μl of alcohol solution (75% EtOH/ 25% benzyl alcohol containing 1% (w/v) lithium dodecyl sulfate) . After vortexing, the nucleic acid was pelleted by a 10 min centrifugation at room temperature in a micro¬ centrifuge. The overlying solution was decanted off, and the pellets rinsed with 0.5 ml of 70% ethanol by gentle inversion of the capped tube three times. Following a brief centrifugation in the microcentrifuge, the 70% ethanol solution was poured off and the pellets dried using vacuum centrifugation. The dried pellets were dissolved in 100 μl of water prior to amplification of the nucleic acid by PCR.

For PCR amplification, 50 μl of each nucleic acid sample prepared above was supplemented with 10 μl of X10 PCR buffer (500 mM KC1, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl₂, 1 mg/ml gelatin), 4.0 μl 25 mM MgCl₂, 0.8 μl 25 mM dNTP, 1.07 μl 467 μg/ml CP514Z prilmer, 0.75 μl 663 μg/ml CP015Z primer, 0.4 μl Taq polymerase, and 33 μl water. After adding a mineral oil overlay, the tubes were capped and placed in a Cetus-Perkin Elmer thermocycler. The thermocycler program consisted of: 1) 1 step cycle of 90 sec at 95°C; 2) 40 step cycles of 30 sec at 95°C, 30 sec at 42°C, and 60 sec at 68^βC (this last step programmed with a 5 sec autoextend); 3) 1 step cycle of 7 min at 68^βC; and 4) a 2°C soak cycle.

Following PCR amplification, the samples were vortexed with 50 μl of Sevag (24:1 chloroform:isoamyl alcohol) to bring the aqueous phase to the top. For analysis, 20 μl aliquots of the amplified samples were incubated in a boiling water bath for 4 min with approxi¬ mately 250,000 cpm of ³P-kinased oligonucleotide CP010 in a total volume of 50 μl containing 150 mM NaCl and 2.5 mM EDTA. The radiolabeled CP010 oligomer hybridizes to an internal portion of the E. pylori amplicon generated by PCR. To visualize the resultant hybrid, the samples were electro- phoresed through a nondenaturing 8% polyacrylamide gel (4 hr at 175 V) , and the gel was subjected to autoradiography overnight at -70^βC with an intensifying screen. Results indicated the presence of . a radiolabeled band of the expected size for those urine samples which had been spiked with 2 x 10³ and 2 x 10* H. pylori. Failure to detect a radiolabeled band in the urine sample spiked with 2 x 10² H. pylori suggests that additional carrier nucleic acid may be required to prevent nonspecific loss of extremely low amounts of target DNA during the extraction process.

B. Blood

Fresh whole blood spiked with a low copy number of an HIV plasmid DNA was successfully extracted using the LiTCA protocol, and the resultant DNA was amplified by PCR and detected by oligonucleotide hybridization. The solutions and protocol were similar to those described in "A." above.

Briefly, each of two 100 μl aliquots of whole blood were lysed with 250 μl of LiTCA lysis solution. Prior to addition of 700 μl of alcohol solution, either 0 or 50 copies of an HIV plasmid DNA were added to the blood lysate. The spiked lysate was then extracted as described for the urine samples above. Care was taken to remove residual lysis-alcohol solution from the DNA pellet before washing the pellet with 70% ethanol.

For PCR amplification, half of each sample (50 μl) was subjected to thermocycling using the protocol described in the Cetus-Perkin Elmer GeneAmplimer HIV kit (N808-0015) . Amplified product was detected as described for the urine samples, except a different radiolabeled probe (i.e., HIV-1 Probe SK19, as described in the Cetus-Perkin Elmer kit) was employed. Following gel electrophoresis and autoradiography, only the extracted blood sample that had been spiked with HIV plasmid DNA gave the expected HIV plasmid band. The 25 copies of HIV plasmid DNA added to the blood lysate and amplified by. PCR produced a very strong signal. Thus, the LiTCA extraction protocol extracted very low copy number DNA from blood samples.

EXAMPLE 9

Isolation of Total Nucleic Acid ithout a centrifigation step

Total nucleic acid (DNA and RNA) was isolated from whole bacterial cells without the use of centrifu¬ gation. Briefly, six aliquots of 1 x 10⁹ cells of Bacteroides gingivalis were each lysed in a 1.5 ml poly¬ propylene microcentrifuge tube with 200 μl of a solution containing 3 M LiTCA, 0.01 M Tris-HCl (pH 7.6), 1 mM LiEDTA and 0.5% (w/v) N-lauroylsarcosine. To each lysate, 400 μl of ethanol were added and the tube was capped and mixed for 30 sec. Nucleic acid was allowed to precipitate for 10 min at ambient temperature.

The suspension of nucleic acid in solution was then wicked through either 5, 8, 10, 50 or 100 micron (mesh opening size) precision woven nylon fabric (Tetko, Inc., Briarcliff Manor, NY). This was accomplished using a 0.75 cm x 0.75 cm square of fabric that was placed over an absorbent pad. The susr. - sion was transferred to the top of the nylon fabric, and . - wicked through the fabric by the action of the absorbent pad. The fabric was rinsed with 100 μl of 70% ethanol, and then each square of fabric was placed in 200 μl of distilled water for 15 min with occasional mixing. As a control, one sample was subjected to centrifugation at 20,000 x g for 10 min to pellet total nucleic acid. The nucleic acid pellet was rinsed once with 70% ethanol, and then dissolved in 200 μl of distilled water. The purity of the nucleic acid preparations was determined by diluting the preparations l:io in distilled water and measuring the optical density of the solutions at 260 and 280 nanometers, as shown in Table 4. Agarose gel electrophoresis of the recovered nucleic acid indicated that both rRNA and chromosomal DNA were isolated intact from the lysed bacterial cells.

TABLE 4

Total Nucleic Acid Extracted With LiTCA and Recovered in the Absence of Centrifugation

The data in Table 4 demonstrate that LiTCA- extracted nucleic acid can be effectively recovered from suspension without the need to perform a centrifugation step. Instead, isolation, purification and recovery of intact nucleic acid is accomplished by retention of precipitated nucleic acid on woven fabric.

Claims

1. A method for improved RNA/DNA hybridization, comprising the steps of: combining RNA and complementary DNA with a trichloroacetate-containing composition, thereby forming a hybridization mixture; incubating the hybridization mixture at a temperature sufficient to optimize the rate of RNA/DNA duplex formation.

2. The method of Claim 1 wherein the RNA is target nucleic acid and the complementary DNA is a signal probe.

3. The method of Claim 2 wherein the complementary DNA further comprises an immobilized capture probe, wherein the capture probe and the signal probe are complementary to non-overlapping target nucleic acid sequences.

4. The method of Claim 1 wherein the complementary DNA is target double strand DNA and the RNA is a signal probe.

5. The method of Claim 4 wherein the RNA further comprises an immobilized capture probe, wherein the capture probe and the signal probe are complementary to non- overlapping target nucleic acid sequences.

6. The method of Claim 1 wherein the trichloroacetate is selected from the group consisting of rubidium trichloroacetate, sodium trichloroacetate, lithium trichloroacetate, potassium trichloroacetate and cesium trichloroacetate.

7. The method of Claim 1 wherein the temperature is about -10^βC to about -l^βC less than the Tm of the RNA/DNA duplex in the trichloroacetate-containing composition.

8. The method of Claim 5 wherein the capture probe or the signal probe is a 2*-0-methyl oligonucleotide.

9. The method of Claim 5 wherein the capture probe and the signal probe are 2*-O-methyl oligonucleotides.

10. A composition for improved RNA/DNA hybridization comprising a trichloroacetate salt in a buffer solution having a pH from about 6 to about 9.

11. The composition of Claim 10 further comprising a detergent selected from the group consisting of sodium dodecyl sulfate, lithium dodecyl sulfate and N- lauroylsarcosine.

12. The composition of Claim 10 further comprising a chelate selected from the group consisting of sodium EDTA, lithium EDTA, sodium EGTA and lithium EGTA.

13. The composition of Claim 10 wherein the trichloroacetate is selected from the group consisting of rubidium trichloroacetate, sodium trichloroacetate, lithium trichloroacetate, potassium trichloroacetate and cesium trichloroacetate.

14. A method for improved extraction of nucleic acid from a biological sample, comprising the steps of: contacting the biological sample with a trihalogenated acetate-containing composition, thereby releasing nucleic acid in a lysate; combining the lysate with an alcohol in an amount sufficient to precipitate the nucleic acid, thereby forming an aqueous phase having precipitated nucleic acid therein; and removing the aqueous phase from the precipitated nucleic acid.

15. The method of Claim 14 wherein the trihalogenated acetate is selected from the group consisting of lithium trichloroacetate, potassium trichloroacetate, sodium trichloroacetate, lithium tribromoacetate, potassium tribromoacetate and sodium tribromoacetate.

16. The method of Claim 14 wherein the alcohol is a mixture of alcohols.

17. The method of Claim 14 wherein the removing step is performed by centrifugation.

18. The method of Claim 14 wherein the removing step is accomplished by retention of the precipitated nucleic acid on a piece of woven fabric.

19. The method of Claim 18 wherein the woven fabric has a homogeneous fiber diameter and is precision woven.

20. The method of Claim 19 wherein the woven fabric is nylon or polyester and has a mesh range of about 1 to about 100 microns.

21. A composition for improved extraction of nucleic acid comprising a trihalogenated acetate in a buffer solution having a pH from about 6 to about 9.

22. The composition of Claim 21 further comprising a detergent selected from the group consisting of sodium dodecyl sulfate, lithium dodecyl sulfate and N- lauroylsarcosine.

23. The composition of Claim 22 further comprising a chelate selected from the group consisting of sodium EDTA, lithium EDTA and sodium EGTA.

24. The composition of Claim 22 wherein the trihalogenated acetate is selected from the group consisting of lithium trichloroacetate, potassium trichloroacetate, sodium trichloroacetate, lithium tribromoacetate, potassium tribromoacetate and sodium tribromoacetate.