CA1230161A

CA1230161A - Dna sequencing

Info

Publication number: CA1230161A
Application number: CA000477173A
Authority: CA
Inventors: Lyle R. Middendorf; John A. Brumbaugh
Original assignee: University of Nebraska
Current assignee: University of Nebraska
Priority date: 1984-03-29
Filing date: 1985-03-21
Publication date: 1987-12-08
Also published as: AU588229B2; AU4002185A; EP0157280A3; EP0157280B1; US6143151A; JPH0743347B2; EP0157280A2; US4729947A; JPS612077A; DE3583516D1; US6143153A

Abstract

DNA SEQUENCING

ABSTRACT OF THE DISCLOSURE
To sequence long strands of DNA, cloned strands having lengths longer than 100 bases are, in one embodi-ment, marked on one end with biotin. These strands are divided into 4 aliquots and each aliquot: (1) is uniquely chemically treated to randomly terminate the strands at the non-biotinylated end at a selected type of base; and (2) is moved continuously by electrophoresis through a different one of four identical channels. In the one embodiment, the strands are randomly terminated at a selected base type and they are moved into avidin, which due to high affinity, combines with the biotin marked ends of shorter strands before the longer strands are fully resolved in the gel.
The avidin is marked with fluorescein, the strands are scanned and the signals are decoded. In another embodiment, the strands are synthesized, with termina-tion at a selected base type and marked either by the above method or by ethidium bromide.

Description

~23~ 6~ ^

DNA SEQUENCING
This invention relates to the sequencing of DNA
strands.
- In one class of techniques for sequencing DNA, identical cloned strands of DNA are marked. The strands are separated into four batches and either individually cleaved at or synthesized to one of the four base types, which are adenine, guanine, cytosine and thymine (hereinafter A, G, C and ~).
The adenine-, guanine-, cytosine- and thymine-cleaved batches are then electrophoresed for separation. ~he rate of electrophoresis indicates the DNA sequence.
In a prior art sequencing technique of this class, the DNA strands are marked with a rad;oactive marker, cleaved at a different base type in each aliquot, and after being separated by electrophoresis, fi]m is exposed to the gel and developed to indicate the sequence of the bands.
The range of lengths and resolution of th;s type of static detection is limited by the size of the apparatus.
In another prior art sequencing technique of this class, single strands are synthesized to a different base type in each aliquot, and the ~3~

strands are marked radioactively for later detection.
It is also known in the prior art to use fluorescent markers for marking proteins and to pulse the fluorescent markers with light to receive an indication of the presence of a particular protein from the fluorescence.
The above prior art techniques for DNA
sequencing have several disadvantages such as: tl) they are relatively slow; ~2) they are at least partly manual; and (3~ they are limited to relatively short stranas of DNA. -In accordance with the invention, a method for sequencing DNA comprises the steps of preparing cloned DNA strands; preparing from the c].oned DNA
strands fragmented DNA strands with random lengths terminating at different ones of the adenine base, guanine base, cytosine base and thymine base and marking the pieces; applying samples of the fragmented DNA strands after terminating at their respective bases to at least one channel of separating apparatus; separating the strands within at least one channel so that the first bands to be moved completely through the channels are fully resolved while the last bands are unresolved in a ~;~3~

continuous process such that at ]east ten percent of the bands are resolved and moved through the channels while the least mobile bands are yet unresolved near the entrance end of the channel; and identifying and recording the time sequence of the bands in the channel and whether the termlntaion is at an adenine base, guanine base, thymine base or cytosine base in the bandO
Advantageously, the step of separating includes the step of separating the strands by gel electrophoresis and more specifically, the step of separating includes the s~ep ~of separating the strands by ~PLC. Further, the step of preparing a first batch may include the step of cleaving the strands at an adenine on pieces ~ith random ]engths.
In one embodiment, four batches of fragments are prepared each batch being terminated at a different one of A, G, C and T bases and the step of separating includes the step of separating each batch in a corresponding channel. ~here is some advantage in including the steps of applying at least one calibration time base source of DNA to at least one channe] interlaced among the four channels. Preferably, the strands terminating at different bases are identified.

~23U~

Advantageously, the step of identifying includes the steps of marking the DNA strands on one end with biotin before separating; attachlng fluorescent markers to the avidin; and moving said bands sequentially from the gel while maintaining each fragment in each channel separate into a means for marking with fluorescent-marked aviden by combining the fluorescent avidin with the biotin end markers.
Moreover, the step of identifying may further include the steps of: moving the bands in sequence through a medium; scanning said bands with laser light having a narrow band wi~th substantially conforming to the optimum adsorption spectrum of the fluorescent markers; pulsing the laser light with pulses of shorter duration than three nanoseconds;
detecting the fluorscent emission from ~he marker across a narrow selective ban2 width conforming substantially to the optimum emission spectrum of the markers through a second period of time; said second period of time beginning at least fifty nanoseconds from the start of its corresponding pulse of laser light and terminati.ng at a time no greater than one hundred fifty nanosecon~s from the start of the pulse of the laser light; and identifying and recording the time sequence of each of the channels so as to indicate the seauence of DNA fragments~
Apparatus for performing the DNA sequencing comprises separating apparatus; said separating apparatus having at least one channel and at least one channel-introducing section adapted to receive DNA fragments terminated at different ones of the four nucleic acid molecules, A, G, C and ~; means for removlng separated bands of the DNA fragments from the end of said separating apparatus prior to the resolution of the last to be r~eso]ved bands; and means for sensing four separate groups of DNA
fragments in accordance with the terminating base.
Advantageously, the separating apparatus is a gel electrophoresis apparatus or capillary chromatographic equipment. The apparatus may include means for marking said DNA fragments prior to their entrance into the electrophoresis gel with biotin; means for attaching a fluorescent marker to avidin; means for marking said DNA fragments af~er they have been electrophoresed through said electrophoresis apparatus with fluorescenated 20 avidin; means for selectively pulsing said markers with light; means for detecting the fluorescence of ~23~

said marker across a time period in which background fluorsecence is reduced; and sai~ means for detecting including means for con~ertlng said fluorescence into electrical signals indicating the time sequence of bands of DNA marked with fluorescence corresponding to each of the DNA
fragments cleaved at different ones of the nucleic acid groups.

One embodiment of apparatus for performing DNA
sequencing comprises: first gel electrophoresis apparatus; said first gel electrophoresis apparatus having at least four separate channel-lntroducing sections each adapted to receive DNA fragments cleaved at different ones of the four nucleic acid molecules, A, G, C and T; and means for electrophoresing separated bands of the DNA
fragments from the end of said electrophoresis apparatus prior to the resolution of the last to be resolved bands.
Advantageously, the apparatus also includes:
means for marking said DNA fragments prior to their entrance into the electrophoresis gel with biotin;

said means for electrophoresing said bands from said gel including means for maintaining four separate groups of DN~ fragments in accordance with the 3L23~

nucleic acid molecu~e at which they were cleaved;
means for marking said DN~ fragments after they have been electroprhoresed through said electrophoresis apparatus with avidin; and means for attaching a flourescent marker to said avidin. There may be included in this embodiment means for selectively pulsing said markers with light, means for detecting the flo~rescence of said marker across a time period in which backgroung flo~rescence is reaucea; and said means for detecting including means for converting said flourescence into electrical signals indicating the time sequence of bands of DNA marked with flourescence corresponding to each of the DNA
fragments cleaved at different ones o~ the nucleic acid groups.
In another embodiment, apparatus for performing DNA sequencing comprises: first gel electrophoresis apparatus; said first gel electrophoresis apparatus inc]uding at least four separate tubes each adapted to receive DNA fragments terminated at differnet ones of the four bases, A, G, C and T; and means for electrophoresing separated bands of the DNA fraqments from the end of said elec~rophoresis apparatus prior to the resolution of the last to be resolYed bands. There may be ~2;~

included in this embodiment means for marking said DNA fragments prior to their entrance into the electrophoresis gel with biotin; said means for electrophoresing said bands from said gel inc]uding means for maintaining four separate groups of DNA
fragments in accordance with the nucleic acid molecule at which they were cleaved; means for marking said DNA fragments after they have been electrophoresed through said electrophoresis apparatus with avidin; and means for attaching a flourescnet marker to said avidin.
The above noted and other features of ~he invention will be better understood from the following detailed description when considered with reference to the accompaning drawings in which:
FIG. 1 is a block diaqram of an embodiment of the invention;
FIG. 2 is a block diagram of another embodiment of the invention;
FIG. 3 is a simplified schematic of a portion of the embodiment of FIGS. 1 and 2;
FIG. 4 is an alternative embodiment of the portion of FIG. 3;

FIG. 5 is another alternative embodiment of the portion of FIG. 3;

~:~3~

FIG. 5 is a block diagram of a portion of the embodiments of FIGS. l and 2;
FIG. 7 is a logical circuit diagram of a portion of the block diagram of FIG. 3; and FIG. 8 is a schematic circuit diagram of a portion of the embodiments of FIGS. l and 2.
In FIG. l, there is shown a block diagram of a DNA sequencing system lO having a biotin labe].ing system ll, a DN~ cleavage system ]2, a separating system 14, a detection and processing sys~em 16 and a source of standard length DMA 18. Biotin labeling takes place before dividing the DNA cl.oned strands into 4 aliquots.
The biotin from any suitable commercia]. source is added to the cloned strands of more than lO0 bases in a container as indicated at 31~ The biotin preparation must be sufficient to mark at least one end of a substantia]. proportion of the DMA fragments with the biotln in a manner known in the art.
Biotin is selected be~ause of its affinity to avidin and because it is not a large molecule, which in the latter case when added to the DNA fragments might substantially dominate the mobility cf the DN~ fragments during electrophoresisO Being a small.
molecul.e, it does not prevent the discrimination ~23~16~

betweer. different DNA fragments within the separating system 14.
Although biotin has been selected as a marker which may be combined later with a larger molecule sush as avidin, other markers may be used. They must have characteristics which enable them to be attached to a DNA fragment and to have a strong affinity to a larger molecule which may be marked with a fluorescein or other suitably detectable material. ~hey must also be of such a size and have such chemical character;stics to not obscure the normal differences in the mobilities between the different fragments due to cleavages at different ones of the adenine, guanine, cytosine ana thymine bases.
In addition, a radioactive marker such as radioactive phosphorus or radioactive sulfur~ radio-active carbon or tritium may be incorporated into the DMA molecules such that after separationr strands are combined with scintillation liquid.
~he DNA cleavage system 12 communicates in four paths and the source of standard length DNA 18 communi-cates in one path within the separating system 14 to permit passage of DNA fragments and standard fragments thereto in separate paths. ~he separating system 14, which sequences strands by separation, communicates with 123~

the detection and processing system 15 which analyzes the fragments by comparison with each other and the standard from the source of standard length DNA 18 to derive information about the DNA sequence of the original fragments.
The DNA c]eavage system 12 incl~des four sources 20A, 20G, 20C, 20T of fragments of the same cloned DNA
strand. This DNA strand is normally greater ~han lO0 bases ln lenqth and is then further cleaved by chemical treatment to provide different lengths of fragments in each of four containers 20A, 20G,-20C and 20T.
In one embodiment, the container 20A contains fragments of DNA strands randomly cleaved by a chemical treatment for A; the container 20G contains fragments of DNA strands randomly cleaved by a chemical treatment for G; container 20C contains fragments of DNA strands randomly cleaved by a chemical treatment for C and container 20T contains fragments of 3NA strands randomly cleaved by a chemical treatment for T. Thus, ldentica~
fragments in each container have been cleaved at different bases of a given base type by the appropriate chem~cal treatment.
The fragments in the containers are respective]y referred to as A-DNA fragments, G-DNA fragments, C-DN~
fragments and T-DNA frag~ents from the containers 20A, S 23~6~

20G, 20C and 20T respectively. These fragments are flowed from the containers 20~/ 20G, 20C and ~OT through corresponding ones of the conduits 22A, 22G, 22C and 22T
into contact with the separating system 14.
The source of standard length DNA 18 includes a source of reference DNA fragments of known but different lengths which are flowed ~hrough a conduit 22S to the separating system 14. These reference fragments have known lengths and therefore their time of movement through the separating system 14 forms a clock source or timing source as explained hereinafter. While in the preferred embodiment the cloned strands of 100 bases are marked with biotin before being divided into four batches, they may be marked instead after dividing into four batches but before the selected chemical treatment.
The separating system 14 includes five electropho-resis channels 26S, 26A~ 26G, 26C and 26T. The electro-phoresis channels 26S, 26A, 26G, 26C and 26~ include in the preferred embodiment, gel electrophoresis apparatus with each path length of gel being identical and having the same field applied across it to move samples continuously through five channels. The ge]s and fields are selected ~o provide a mobility to DNA strands that does not differ from channel to channel by more than 5 in velocity. In addition, the field may be varied over ~z~

time to enhance the speed of larger molecules after smaller molecules have been detected, as well as to adjust the velocities in each channel based on feedback from the clock channel to compensate for differences in each channel such that the mobilities in each channe]
are within the accuracy required to maintain synchronism among the channels.
Preferably the gels are of the same materials~
chemical derivatives and lengths and the electric fields are within 5~ of the intermediates of each other in each channel. ~owever, more than one reference channel can be used such that a reference channel is adjacent to a sample channel in order to minimize the requirements for uniformity of DNA movement in the gel for all channels.
The electrophoresis channel 26S receives fragments of known length DNA marked with biotin and moves them through the gel. Similarly, each of the electrophoresis channels 26A, 26G, 26C and 26T receives biotin-labeled fragments from the cleavage system 20A, 20G, 20C and 20T
and moves them in sequence through the sample elec-trophoresis channels, with each being moved in accor-dance with its mobility under a field ident;cal to that of the reference electrophoresis channel 26S.
To provide information concerning the DNA sequence, the detection and processing system 16 includes flve ~23~16~

avidin sources 30S, 30A, 30G, 30C and 30T; five detection systems 32S, 32A, 32C-, 32C and 32T and a correlation system 34. Each of the avidin sources 30S, 30A, 30G, 30C and 30T is connected to the detectinq systems 32S, 32A, 32G, 32C and 32T. Each of the outputs from corresponding ones of the electrophoresis channels 26S, 25A, 26G, 26C and 26T within the separating system 14 is connected to a corresponding one of the detection systems 32S, 32A, 32G, 32C and 32T. In the detection system, avidin with ~luorescent markers attached and DNA fragments are combined to provide avidin marked DNA
fragments with fluorescent markers attached to the avidin to a sample volume within the detection system for the detection of bands indicating the presence or absence of the fragments, which over time relates to their length.
The output from each of the detection systems 32S, 32A, 32G, 32C and 32T are el.ectrically ~onnected through conductors to the correlation system 34 which may be a microprocessor system for correlating the ;nformation from each of the detection systems to pro-v1de information concerning the DNA sequence.
The avidin sources 30S, 30A, 30G, 30C and 30T each contain avidin purchased from kno~n suppliers, with each avidin molecule in the pre~erred embodiment combine~

~3V~.61 with three fluorescein moleules. The avidin ~ources are arranged to contact the ~NA fragments and may be in a section of gel placed adjacent to the electrophoresis channel. In this use, this section of the gel should have a p~I of approximately 8 to avoid movement of the three fluorescein-marked avidin by electrophoresis. when the biotinylated DN~ strands reach the section of gel that has a p~ of 8, they will pick up the fluoresceinated avidin which moves very slowly or is stationa~y in this section of the ge].
To prepare the second section of gel with fluoresceinated avidin, the fluoresceinated avidin may be electrophoresed from the exit end of the channel inwardly. In this embQdiment, it moves in this direction slowly because its pI is slightly higher than the p~ of the second section of gel. Alternatively, it may be mixed in gel.
Because the fluorescein-avidin-biotin-DNA complex molecule is acidic in the pn 8 gel, it will continue to move out of this section of the gel where it is then passed to a sample volume within the detection system by an eluant. The sequences of separation determine~
before the attachment of avidin are maintained and not substantially altered. In the alternative, the bands of DNA fragments may be e]ectrophoresed ;nto a more mobile ~L~3(~

liquid containing fluorescein marked avidin for combina-tion with the avidin. The avi.din binds se1.ectively to the biotin attached to the ends of the DNA fragments and unreacted fluoresceinated avidin is separated from the fluorescein-avidin-biotin-DNA complex by standard techniques such as chromatography.
The detection systems each include an optical system for detecting the presence or absence of bands and converting the detection of them to electrical sig~
nals which are applied electrically to the correlation system 34 indicat;nq the sequence of the fragments with respect to both the standard fragments from the source o standard length DNA 18 and the A, G, C and T frag-ments from the containers 20A, 20G, 20C and 20 respectively.
In FIG. 2, there is shown a simplified block diagram of another embodiment of chromatographic appara-tus A10. This apparatus is similar to the chromatographic apparatus 10 of FIG. 1 and the compo-nents are identified in a similar manner with the reference numbers being prefixed by the letter A.
In this embodiment, insteafl of the containers for DNA and chemical treatment for A, G, C and T of the embodiment of DNA sequencing system 10 shown at 20A, 20G, 2QC and 20~ in ~IG. 1, the chromatographic ~23~3~6~

apparatus 10 includes containers for treatment of the DNA in accordance with the method of Sanger described by F. Sanger, S. Nicklen and ~.R. Coulson, ~DNA Sequencing with Chain-Terminating Inhibiters," Proceedings of the National Academy of Science, USA, Vol. 74, No. 12, 5463-5467~ 1977, indicated in the embodiment AlO of FIG. 2 at A20A, A20G, A20C and ~20T shown as a group generally at A12.
In this method, the strands are separated and used as ~emplates to synthesize DNA wi~h synthesis terminating at given base types A, G, C or T in a r~ndom manner so as to obtain a plurality of different molecular weight strands. The limited synthesis is obtained by uslng nucleotides which will terminate synthesis and is performed in separate containers, one of which has the special A nucleotide, another the special G nucleotide, another the special C nucleotide and another the special ~ nucleotide. These special nucleotides may be dideoxy nucleotides or marked nucleo~ides, both o which would terminate synthesis.
So, each or the four batches will be ~erminated at a different one of the types of bases A, G, C and T
randomly.
In this embodiment, the fragments may be marked by biotin at one end in the manner shown in FIG. 1.

~8 lg ~owever, in the preferred embodiment of FIG. 2, lnstead of labeling with biotin, thle fragments are labeled by an inverted complementary repeat of DNA as shown at A]l before bein~ applied to the channels ;ndicated at A12 in FIG. 2. The desi~n of in~erted complementary repea~
takes advantage of the pro~ess of designlnq small DNA
frasments known as oligonucleotides. This process is widely described in the literature as we~l as such patents as Phosphoramidite Components and Processes (U.S~ pa~ent 4,415~732), After the electrophoresis, the inverted complemen-tary repeat forms a hairpin from a pa]indrome of duplex DNA, which is then combined w;th ethidium bromide and detected by the detection system using a wavelength of 7ight appropriate to the intercalated ethidium bromide rather than wavelengths of ~ight appropriate to the fluorescein marking. If one uses highly æen itive detection techniques, the inverted repeat would not be used and detection would be accompllshed either by 1 20 sensing ethidium bromide that intercalated between por-¦ tions of the unknown DNA that happened to form duplex DNA, or by ethidium bromide that a~tached to single stranded DNA~ or by the inherent fluorescence of the DNA
itself. If one used radioactive markers, detectlon ~23V~

would be accomplished by sensing light given off.by the combination of the radioactive marker and scintillation fluid.
In FIG. 3, there is shown a separating system 26A
which includes a slab of gel 27 as known in the art with five sa~ple dispensing tubes in~ioated generally at 29A terminating in alignea slots 51 in the gel. 27 on one end, with such slots in contact with a negative potential buffer well. 29 having a negative electrode 47A, and flve exit tubes at the other end located at 31A terminating in apert~lres in the gel 27, as well as a positive potential buffer well 31 having a positive electrode A53. The material to be electrophoresed is inserted into slots 29A and due to the field across the gel 27 ~oves from top to bottom in the gel and into the appropriate corresponding exit tubes of the group 31A.
The gel slab 27 has glass plates 27A and 27~ on either side to confine the sample and gel. Buffer fluid from the buffer well 31 is pumped at right angles to the gel 27 from a source at A57 by pumps connected to tubes 31~
to pull fluid therethrough. The buffer fluid p;cks up any DNA that is electrophoresed into the exit holes 3~A
and makes its way to sensing equipment ~o be described hereinafter or to provide communication with other gel

2~

3(3~

slabs for further electrophores;s of the DNA strands being electrophoresed from the slab 27.-In FIG. 4, there is shown another embodiment B26Aof gel electrophoresis having a negative-potential buffer for the A channel indicated genera]ly at B29A, a gel electrophoresis channel for ~ terminated DNA
indicated at B27A and a positive potential buffer for the channel îndicated at B31A. This embodiment is intended to provide a thin cylindrical gel for each channel so as to permit easier temperature control and thus alleviate changes in migration rates by different temperatures such as may occur across the slab 27 (FIG~
3~. In addition, the field could be adjusted independently for each channel to maintain proper synchronization.
For this purpose, the channel B27A includes a 0.5 to a 1 millimeter inner diameter pyrex column such as a chromatographic column indicated at 33 with a gel inside of it. The gel is prepared by inserting it in the column while warm and permitting it to harden. The column 33 is of sufficient length to separate the DNA.
Fluoresceinated avidin may be initially e~ectrophsresed upward ~rom its exit end shown at 35 to meet with DNA
entering from its entrance end shown at 37 in the embodiment wh-ch uses biotin-avidin-fluorescein as a ~23~

marker. mhe column may be temperature controlled by a conventional chromatographlc temperature controlling apparatus 39 which is a glass casing about the column which receives a liquid at one end such as at 41 and removes it at 43.
At one end of the channel s27A is the buffer B29A
adapted to provide a buffer solution in a p~exiglas surrounding cup shaped container 45, which buffer ex-tends over the entering end 37 and contains within it a negative voltage electrode 47.

At the exit end, there is similar].y mounted a buffer compartment 51 containing buffer which is grounded by an electrode 53 and emerses in buffer the exit end 35 of the gel column B27A. It may be made of plexiglass and may be shaped with a reducing oriface ending in a micro-orifare 55 at its lower end to permit the flow of buffer therethrough containing DNA which emerged from exit end 35 for detecting with a chopped light source as described above. To supply new buffer, a buffer reservoir 57 is connected through a pump 59 to the top of the buffer 51, with the fl.ow rAtes being designed to prevent a vortex near the exit end 35 and to permit a flow rate sufficient for optimum signal-to-~oise ratio of buffer~ Another embodiment would ~Z3~

transfer the flowing buffer-DNA solution to a f]ow-through cell for detection in a spectrofluorometer or specifically designed HPLC fluorescence detection.
In one embodiment, the buffer may include the ethidium bromide for exciting 300 or 390 nanometers and detecting at 590 nanometers.
In FIG. 5, there is shown still another embodiment C26A which may be substituted for the column 33 and gel and includes capillary columns 61, 63 and 65 as commonly used in capillary electrophoresis. These columns may be filled with buffer solution rather than a gel and be used for electrophoresis. In such a case, several capil]aries may be used as a substitute for one column of the embodiment of FIG. 4. Thus, the same band of A, G, C or T type bases might flow through several parallel bundles of capillaries, or they might flow through on]y one capillary per type of base.
The separation path such as gel channels or capillary ~ube length should be no tonger than two meters for a range of lengths of DNA from 50 to 10,000 or more bases. However, as the range of DNA ]engths increase, the time required increases. Also, the time required for each separation is in the range of from 1/2 second to 5 minutes for each added base of length separation.

~23~
2~

In FIG. 6 there is shown a b],ock diagram of the detection system 32~. The detection systems 32S, 32G, 32C and 32T are substantially identical to the detection system 32A and sc only the system 32~ will be described in detail herein. The detec~ion system 32A includes an electrophoresis channel 42, a sample volume 43, a light source 44 and an optical detection system 46.
In one embodiment avidin marked with fluorescein in the ~luorescenated-avidin source flows into the gel which receives A type terminated strands from channel 26A on conduit 48 and i5 attached to the biotinylated DNA fragments. Actually, the electrophoresis channel 42 may be a gel section positioned at the end of the electrophoresis channel 26A for continuous electro-phoresing. After the fluoresceinated avidin is attached to the biotinylated DNA in the electrophoresis channel 42, the complex molecule is eluted out of the electrophoresis channel 42 into the sample volume.
'In another embodiment, the DNA may be marked by a palidrome described above and the detection system would then utilize a different wavelength of light and wou],d not require the fluorescenated-avidin source 40 but rather an ethidium bromide source 40A.
The sample volume 43 is irradiated by the light source 44. Li~ht from the light source 44 is detected ~36~

and converted to electrical signals by the optical detection system 46 for application through the eondue-tor 50A to the correlation system 34 tFIG. '). In one embodiment, the fluorescenated-avidin source 40 contains a fluorescent marker having a period of fluorescenee sufficiently long compared to background fluorescenee in the DNA and associated materials to permit significant separation of the signal from the fluoreseence. In this embodiment, the decay lifet;me of the fluoreseent marker should be at least ten times the duration of the baekground fluorescenee whieh baekground fluoreseenee is expressed in the form of noise in a detected signal.
Some known appropriate fluoreseent markers are:
(1) rare-earth organo eomplexes, eonsisting of rare earth bound to organie eompounds with ~he resulting eomplex having the desired properties, sueh as euro-pium, benzoylaeetonate and europium benzoyltrifluoro-acetonate, as discussed by S.I. Weissman in the Journal of Chemical Physics, vol. 10, page 214-217, 1942; (2) pyrenebuterate, as discussed by Knopp and Weber in the Journal of Biological Chem_ try, vol. 242, p. 1353 _ (1963) and vol. 244, p. 3609 (1969); (3) fluoreseein isothiocyanate (FITC); (4) rhodamine ~Z3~61 isothiocyanate ~RITC~; (5~ tetramethylrhodamine isothio-cyanate (TRITC); (63 phycoerythrin; and (7~ their analogs and substituted derivatives. Such materials are available commercially such as from ~esearch Organics in Cleveland, Ohio. In the preferred embodiment, fluorescein avidin DCS iS purchased Erom vector Laboratories, Inc., 1429 Rollins Road, Burlingame, CA
94010.
The light source 44 includes a pulsed light source 52 and a modulator 54. The pulsed light source 52 is selected to emit light within the absorbance spectrum of the fluorescent marker. Since different f~uorescent markers may be used, this frequency differs from fluorescent marker to fluorescent marker. Moreover, in one embodiment, the modulator 54 controls the pulsed light source 52 to select intervals between pu]ses, the intervals being provided to permit the decay of fluores-cent light from the background fluorescent material, during which time the fluorescent light from the bound markers is measured.
These time periods between pulses are suffi-ciently long to encompass the entire delay period. This is done because the delay period of the attached fluore-scent marker is relatively lonq compared to background noi~e fluorescence and so a period of time may pas.s ~6 ~L23~

before the measurement is made by the optical detection system 46. Typically, the pulse of liqht has a duration of approximately three nanoseconds and the background fluorescence decay lasts for approximately ten nano-seconds while the fluorescent marker attached to the avidin has a decay lifetime of 100 nanoseconds.
Typically, the optical detection system 46 begins reading at approximately 50 nanoseconds after the ini-tia~ion of the excitation p~lse from the laser and continues for approximate]y 150 nanoseconds until 200 nanoseconds after the initiation of the three nanosecond pulse. Although in the preferred embodiment, a pulsed laser light source 52 is ut;lized, a broad band light source combined with filters or a monochrometer may be utilized to provide the narrow band in the absorption spectrum of the marker.
Another embodiment uses an electro-optic modulator which modulates a continuous light SQUrCe at a frequency typical3y at 10 khz, with essentially 100~ depth of modulation and 50~ duty cycle. A pulse generator pro-vides a signal both to the modulator via a driver and to a lock-in amplifier as a reference signal. Another embodiment uses a continuous light source with no modulations.

123~16~L

To detect the bands in the electrophoresis gel of the electrophoresis channel 42 indicating particular DNA
fragments, the optical detection system 46 includes certain viewing optics 60, a filter 62, and an optical detection system 64. The filter 62 selects the fre-quency of light transmitted thro~lgh it by the viewing optics 60 which focuses the ligh~ onto the optical detectlon system 64. The optical detection system 64 is electrically connected ~o the modulator 54 to gate an electrical signal to the conductor 50A indicating the presence or absence of a band of DNA fragments in the electrophoresis channel 42.
The filter 62 in the preferred embodiment includes an interference filter having a pass band corresponding to the high emission spectrum of the fluorescent marker.
Such filters are known in the art and may be purchase~
from commercial sources with bands to correspond to common emission bands of fluorescent markers. In addi~
tion, there may be long-wave~ength-passing interference filters and/or colored glass filters. Another embodiment uses a monochrometer instead of a filter.
The viewing optics 60 consists of a lens system positioned in juxtaposition with filter 62 to focus light onto the optica~ detection system 64. ~t may be ~3~

any conventiona] optical system, and the optical detec-tion system 64 should include a semiconductor detector or a pho~omultiplier tube, such as the Model ~MI 9798A
made by EMI Gencon, Plainview, New York.
In the first embodiment~ the output of the photo-multiplier or semiconductor is gated in response ~o the signals from the modulator 54 to occur after a time delay after each pulse from the pulsed laser light source 52. For example, a time delay may be included before the electrical signal is applied to an amplifier and thus provide an electrical signa] to the conductor 50A or to an amplifier, the output of which is electrically connected to the conductor 50A~ In the preferred embodiment, the time delay is 50 microseconds and the gate or amplifier is maintained open by a mono-stable multivibrator for approximately 150 nanoseconds.
In the second embodiment, the square wave output of a modulator is compared with the signal from the detector using a lock-in amplifier. In a third embodiment, no modulation is performed.
In FIG. 7 there is shown a block diagram of the correlation system 34 having a standard channel input circuit 70S, a gating sys~em 72, a decoder 74, a memory 76 and a read-out system 78. ~he standard channel input circuit 70S is electrically connect2d to 3~

the OR gate 74S which is electrica]ly connected to the other channels A, G, C and T and to the gating system 72 which receives channel input signals from each of the channels A, G, C, and T s;milar to that of channel 70S.
The gate 74S is electrica1ly connected to the memory 76 which receive signals from gate 74S inaicating the presence of ~NA fragments in the particular one of the nucleic acid bases or in the standard channel. The memory 76 is electrically connected to the read-out system 78 to print out the sequence.
The standard channel input ~ircuit 70s includes a pulse shaper 82A, a binary counter 84S, a time de],ay 94S
of the clock 80 and a latch 86S with the input of the pulse shaper 82S being electrically connected to a con-auctor 50s and its output being connected to OR gate 74S
through time delay 94S and to the binary counter 84S.
The ouput of the binary counter 84S is connected to the latch 86 to provide a time increment signal ~o the latch 86, the output of which is applied to one of the inputs of memory 76 when triggered by A signa] from OR
gate 74S. The conductor 50S corresponds to conductors 50A, 50G, 50C and 50T ~FIG. 2) except that conductor 50S is the output for the standard clock channel rather than for adenine. quanine, cytosine or thym1ne.

~.230~

The latch 86 and the decoder 74 are pulsed by a signal from the ga~e 74S to write into the memory 76 for recording with a distinctive signal indicating a clock timing pulse which is later printed to indicate the time that particular DNA segments have been received and detected in the detection system 32A, 32G, 32C and 32T (FIG. 1). The binary counter ~4S receives clock pulses from clock 80 to which it is connected and thus contains a binary signal representing tlme for applica-tion to the latch 86.
The switching circuit 72 includes a dec~der 74 which is electrically connected to four inputs from channels 70A, 70G, 70C and 70T respectively, for receiving signals indicating the presence of types A, G, C, and T, fragments as they appear on input conductors 50A, 50G, 50C and 50T, (of FIG. 1 and F~G. 2). The signals on conductors 50A, 50G, 50C, and 50T are each applied to respective ones of the pulse shapers 82A, 82G, 82C, and 82~, the outputs of which are e].ectrically connected through corresponding ones of the conductors 92A, 92G, 92C, and 92T to different inpu~s of the decoder 74 and to inputs of the O~ gate 74S, so that the decoder 74 receives signals indicating the presence of a DNA fragment for application to the memory 76 upon receiving a signal on conduc~or 90S from the OR gate 3L23~ 6~

74S. The OR gate 74S applies such a signal whenreceivins a calibration signal from the channel 70S or when receiving a signal from any one of the channels 70A, 70G, 70C, and 70T, so that the memory 96 receives calibration signals or signals indicatins DNA for reading out, after a de~ay within the memory 7~, to the readout system 78.
The OR gate 74S recei.ves its calibration signal from channel 70S after a delay imparted in ~he delay llne 74S, but does not have such a corresponding delay in channel 70A, 70G, 70C, and 70T. ~owever, a similar delay is within encoder 74 to be described he~einafter so that the appearance of DNA fragments wi~l be sent to the memory 76 in a time frame corresponding to that of the calibration signals from channel 70S. The output of the decoder 74 is electrically connected to the memory 76 through a conductor 100.
In FIG. 8, there is shown a schematic circuit diagram of the decoder 74 having a delay line 94, an OR
gate 102 and a plurality of coding channels 74A, 74G, 74C and 74T to respectively indicate fra~ments termi-nating with the bases, adenine, guanine, cytoslne and thymine respectively.
The channel 74A includes AN3 gate 106, having its inputs electrically connected to conductor 92A and ~Z3~61 90A to receive on conductor 90~ a clock signal from the OR gate 7~S (FIC,. 7) and on its other input a signal indicating the presence of an adenine ter~inated frag-ment on conductor 92A.
Channel 74G includes AND gate 108, AND gate 110 and delay line 112. Conductor 92~ indicating a guanine terminated strand is electrically connected to the in~
puts of AND gate 108 and 110. The output of AND gate 108 is connected to one of the inputs of OR gate 102 and the output of AND gate 110 is electrica~.ly connected through delay line 112 to the input of OR gate 102 to provide two pulses in succession to gate 102. Thus, channel 74A applies one out pulse from the output of AND gate 106 to one of the inputs of OR gate 102, whereas channel 74G applies two pulses. In either case, the sequence of pulses indicates the presence of a particular one of the types of DNA fragments A or G.
Similarly, the channel 74C includes AND gates 3~4, 116 and 118, each having one of its two inputs electrically connected to conductor 92C and 90C and the channel 74T includes AND gates 120, 122, 124 and 126, each having one of its inputs electrically connected to conductor 92T and the other connected to 90T. The output from an AND gate 74C is electrically connected to an input of OR gate 102, the output of AND gate llÇ

6 ~

is electrically connected through a delay 128 to the input of OR gate 102, and the output of AND gate 11 is electrically connected through a delay 130 longer than the delay 128 to an input of the OR gate 106. With this arrangement, the presence of a DNA strand termina-ting ~tith cytosine results in three pulses to the OR
gate 102.
The output of AND gate 120 is electrically connected to an input of the OR gate 102, the ou~put of the AND gate 122 is electrically csnnected through a delay 132 to an input of the OR gate 102, the output of AND gate 124 is electrically connected through a delay 134 longer than the delay 132 to an input of the OR gate 102 and the output of AND gate 126 is electrically connected through a delay 136 longer than the delay 134 to an input of the OR gate 102. In this manner, the presence of a thymlne-terminated fragment results in four signals in series to the inputs of OR gate 102.
The out gate of OR gate 102 is applied through a de~ay 94 with a similar time delay as the delay 94S (FIG. 7~
to the output conductor 100 so as to provide a coded signal indicating the presence of a particular DNA group to the memory 76 ~F~G. 7) coordinated with a time signal.

~23~

In the operation of sequencing DNA, DNA strands with bases above 100 in number are first marked with biotin, separated in accordance to the size of the fragment and then marked with avidin marked with one or more fluorescent molecules. The bands are then detected by light with the read-out in the emission spectrum taking place a sufficient amount of time after excitation in the emission spectrum to screen against noise.
To mark DNA fragments with biotin, cloned strands are prepared and cleaved into frayments after which they are first marked wi~h biotin and then divided into four aliquots. A standard source of DN~ strands also marked with biotin and having different rates of migration under electrophoresis forms additional calibration batches. ~he four batches are each individual]y, randomly cleaved by a chemical treatment for a different one of adenine, guanine, cytosine and thymine bases. In the alternative, strands may be separate~ and used as templates for synthesizing randomly to a selected base tvpe. In either case, the strands may be marked with biotin, marked with an inverted complementary sequence of DNA, marked with a radioactive marker or left unmarked.

~23~3~6~

To separate the fragments, the biotin marked frag-ments including any standard ladder source are each individually electrophoresced through gel in different channels or in different columns. The gel ana the field must be uniform, although reference channels reduce uniformity requirements, and when a sing]e slab is used to migrate several different samp]es, the channe]s must be kept separate and be centered sufficiently around the field so that the potentia7 for causing them to migrate is uniform. Preferab]y, the p~ of the gel for separa-tion is 7.5-80 The DNA fragments separate ;n accordance with their length during electrophoresis. Thus, the fastest migrating fraction is the fragment which is cut or synthesized to the first base closest to the marked end of the strand and, since the channels are separate, it is known which base A, G, C or T is the first one in the sequence from the channel.
The next band in time in the gel is the next clea-vage point which is one base longer than the first one since it encompasses both the first base and the second one from the biotinylated end of the DNA strand.
Similarly, the third fragment to for~ a band during electrophoresis will encompass the first three base units and so on.

~Z3V:~6~

Because a large number of bases are used, there is a larger number of cleavage points and the density of fragments in each band is relatively low. Thus, the gel and the Eield must be selected to provide a band of sufficient width, high enough density and ade~uate sepa-ration for detection. The gel slab is sufficienty long such that the first bands to be moved completely through the gel are fully resolved while the last bands are unresolved in a continuous process. More specifically, at least 10 percent of the bands are resolved and electrophoresed through ~he gel while the least mobile bands are yet unresolved near the entrance end oE ~he gel.
To provide light amplification for measurement of the low density bands in one embodiment, the bands are electrophoresced into a region where they are mixed with avidin marked with one or more fluorescent molecules. Because avidin is a large molecule and strongly attracted to the biotin, the DNA fragments in each band, as they are moved into a fluorescent-marked avidin region, are marked with avidin~ ~fter being marked with avidin such as indicated at 32S, 32A, 32G, 32C and 32T (FIG. 1), they are each moved through a detection system such as the one illustrated in FIG~ 2.

~23v~e~l Because the avidin molecules are large, a number of fluorescent markers are at~ached to the same avidin molecule thus providing adequa~e detection. The fluore-scent-marked fragments are then moved into the samp]e volume within the detection system where they maintain their relative order. The movement must be sufficiently rap;d in the gel so that minimum resolution is lost.
The bands are eluted into the sample volume where individual light sources apply pulsed or chopped light within the optimum absorption spectrum of the f]uore-scent marker or ethidium bromide marker in the second embodiment of approximately three nanoseconds duration.
The light is sensed by a detector approximately fifty nanoseconds after the beginning of the three nanosecond pulse of light and the resulting electrical signal is amplified. This light is detected and corre~ated to provide the sequence of DNA in accordance with the channel as indicated by a detector at the end of each of the detection systems.
As can be understood from the above description, the DNA sequencing system of this invention enables continuous sequencing and thus may handle in a con-tinuous, automatic manner, a large number of bases.
This is accomplished by the combination of continuous electrophorescing with the amplification provided by 3~

the avidin attachment at the end of the first separa~
tion.
Although a preferred embodiment of the invention has been described with some particularity, many modi-fications and variations are possible in the preferred embodiment within the light of the above description.
Accordingly, within the scope of the appended claims, the invention may be practiced other ~han as specifi-cally described.

Claims

The embodiments of the invention for which an exclusive right or privilege is claimed are:

1. A method for sequencing DNA comprising the steps of:
preparing cloned DNA strands;
preparing from the cloned DNA strands fragmented DNA strands with random lengths terminating at different ones of the adenine base, guanine base, cytosine base and thymine base and marking the pieces;
applying samples of the fragmented DNA strands after terminating at their respective bases to at least one channel of separating aparatus;
separating the strands within at least one channel so that the first bands to be moved completely through the channels are fully resolved while the last bands are unresolved in a continuous process such that at least ten percent of the bands are resolved and moved through the channels while the least mobile bands are yet unresolved near the entrance end of the channel; and identifying and recording the time sequence of the bands in the channel and whether the termination is at an adenine base, guanine base, thymine base or cytosine base in the band.

2. A method according to claim 1 in which the step of separating includes the step of separating the strands by gel electrophoresis.

3. A method according to claim 1 in which the step of separating includes the step of separating the strands by HPLC.

4. A method according to any of claims 1 through 3 in which the step of preparing a first batch includes the step of cleaving the strands at an adenine on pieces with random lengths.

5. A method according to any of claims 1 through 3 further including the step of preparing four batchs of fragments each batch being terminated at a different one of A, G, C and T bases and the step of separating includes the step of separating each batch in a corres-ponding channel.

6. A method according to any of claims 1 through 3 including the steps of applying at least one calibration time base source of DNA to at least one channel.

7. A method according to any of claims 1 through 3 further including the step of identifying strands terminating at different bases.

8. A method according to any of claims 1 through 3 in which the step of identifying includes the steps of:
marking the DNA strands on one end with biotin before separating;
attaching fluorescent markers to the avidin; and moving said bands sequentially from the gel while maintaining each fragment in each channel separate into a means for marking with fluorescent-marked aviden by combining the fluorescent avidin with the biotin end markers.

9. A method according to any of claims 1 through 3 in which the step of identifying further includes the steps of:
moving the bands in sequence through a medium;
scanning said bands with laser light having a nar-row band width substantially conforming to the optimum adsorption spectrum of the fluorescent markers;
pulsing the laser light with pulses of shorter duration than three nanoseconds;

detecting the fluorescent emission from the marker across a narrow selective band width conforming substan-tially to the optimum emission spectrum of the markers through a second period of time;
said second period of time beginning at least fifty nanoseconds from the start of its corresponding pulse of laser light and terminating at a time no greater than one hundred fifty nanoseconds from the start of the pulse of the laser light; and identifying and recording the time sequence of each of the channels so as to indicate the sequence of DNA
fragments.

10. Apparatus for performing DNA sequencing comprising:
separating apparatus;
said separating apparatus having at least one channel and at least one channel-introducing section adapted to receive DNA fragments terminated at different ones of the four nucleic acid molecules, A, G, C and T;
means for removing separated bands of the DNA
fragments from the end of said separating apparatus prior to the resolution of the last to be resolved bands; and means for sensing four separate groups of DNA
fragments in accordance with the terminating base.

11. Apparatus in accordance with claim 10 in which said separating apparatus is a gel electrophoresis apparatus.

12. Apparatus in accordance with claim 10 in which said separating apparatus is capillary chromatographic equipment.

13. Apparatus according to any of claims 10 through 12 further including:
means for marking said DNA fragments prior to their entrance into the electrophoresis gel with biotin;
means for attaching a fluorescent marker to avidin;
means for marking said DNA fragments after they have been electrophoresed through said electrophoresis apparatus with fluorescenated avidin;
means for selectively pulsing said markers with light;
means for detecting the fluorescence of said marker across a time period in which background fluorescence is reduced; and said means for detecting including means for converting said fluorescence into electrical signals indicating the time sequence of bands of DNA marked with fluorescence corresponding to each of the DNA fragments cleaved at different ones of the nucleic acid groups.

14. Apparatus for performing DNA sequencing comprising:
first gel electrophoresis apparatus;
said first gel electrophoresis apparatus having at least four separate channel-introducing sections each adapted to receive DNA fragments cleaved at different ones of the four nucleic acid molecules, A, G, C and T;
and means for electrophoresing separated bands of the DNA fragments from the end of said electrophoresis apparatus prior to the resolution of the last to be resolved bands.

15. Apparatus according to claim 14 further comprising:
means for marking said DNA fragments prior to their entrance into the electrophoresis gel with biotin;
said means for electrophoresing said bands from said gel including means for maintaining four separate groups of DNA fragments in accordance with the nucleic acid molecule at which they were cleaved;
means for marking said DNA fragments after they have been electrophoresed through said electrophoresis apparatus with avidin; and means for attaching a fluorescent marker to said avidin.

16. Apparatus according to claim 15 further including:
means for selectively pulsing said markers with light;
means for detecting the fluorescence of said marker across a time period in which background fluorescence is reduced; and said means for detecting including means for converting said fluorescence into electrical signals indicating the time sequence of bands of DNA marked with fluorescence corresponding to each of the DNA fragments cleaved at different ones of the nucleic acid groups.

17. Apparatus for performing DNA sequencing comprising:
first gel electrophoresis apparatus;

said first gel electrophoresis apparatus including at least four separate tubes each adapted to receive DNA
fragments terminated at different ones of the four bases, A, G, C and T; and means for electrophoresing separated bands of the DNA fragments from the end of said electrophoresis apparatus prior to the resolution of the last to be resolved bands.

18. Apparatus according to claim 17 further comprising:
means for marking said DNA fragments prior to their entrance into the electrophoresis gel with biotin;
said means for electrophoresing said bands from said gel including means for maintaining four separate groups of DNA fragments in accordance with the nucleic acid molecule at which they were cleaved;
means for marking said DNA fragments after they have been electrophoresed through said electrophoresis apparatus with avidin; and means for attaching a fluorescent marker to said avidin.