United States Patent c19J
`Cheeseman
`
`[54] METHOD FOR SEQUENCING
`POLYNUCLEOTIDES
`Inventor: Peter C. Cheeseman, Palo Alto, Calif.
`[75]
`[73] Assignee: Beckman Instruments, Inc.,
`Fullerton, Calif.
`[21] Appl. No.: 661,750
`Feb. 27, 1991
`[22] Filed:
`
`[51]
`
`[56]
`
`Related U.S. Application Data
`[63] Continuation of Ser. No. 393,586, Aug. 14, 1989, aban(cid:173)
`doned.
`Int. Cl.5 ........................ C12Q 1/68; C12P 19/34;
`C07H 21/04
`[52] U.S. Cl ...................................... 435/6; 536/24.33;
`536/25.3; 935/77; 935/78
`[58] Field of Search .............. 435/6, 91; 536/27, 25.3,
`536/24.33; 935/77, 78
`References Cited
`U.S. PATENT DOCUMENTS
`5,001,051 3/1991 Miller et al ............................. 435/6
`FOREIGN PATENT DOCUMENTS
`8909282 10/1989 PCT Int'l Appl. ..................... 435/6
`OTHER PUBLICATIONS
`"A Photoinduced Cleavage of DNA Useful for Determin(cid:173)
`ing T Residues", A. Simoncsits et al., Nucleic Acids
`Research, vol. 10/No. 24 (1982) pp. 7959-7965.
`"Molecular Biology", Second Edition, D. Frcifelder, pp.
`85-86.
`"Primer-Directed Enzymatic Amplification of DNA with
`a Thermostable DNA Polymerase", R. Saiki et al, Sci(cid:173)
`ence, vol. 239, pp. 487-491.
`"Light-Directed, Spatially Addressable Parallel Chemical
`Synthesis", S. Fodor et al, Science, vol. 251, pp.
`767-773.
`"Photoremovable Protecting Groups in Organic Synthe(cid:173)
`sis", V. N. Rajasekharan, Synthesis, 1980, pp. 1-26.
`"Organic Chemistry of Nucleic Acids", N. Kochetkov et
`al, Part B, Plenum Press (1972) Chapter 9, pp. 449-476.
`"Automated DNA Sequencing: Ultrasensitive Detection of
`Fluorescent Bands During Electrophoresis", W. Ansorge
`
`I IIIII IIIIIIII Ill lllll lllll lllll lllll lllll lllll lllll lllll llllll Ill lllll llll
`US005302509A
`5,302,509
`[11] Patent Number:
`[45] Date of Patent: Apr. 12, 1994
`
`ct al., Nucleic Acids Research, vol. 15/No. 11 (1987)
`pp. 4593"4602.
`"Solid Phase DNA Sequencing Using the Biotin-Avidin
`System", S. Stahl et al, Nucleic Acids Research, vol.
`16/No. 7 (1988) pp. 3025-3038.
`"Automated DNA Sequence Analysis", C. Connell ct al.,
`BioFcature, vol. 5, No. 4 (1987) pp. 342-348.
`"Molecular Biology and Biotechnology", J. Walker et al.,
`Second Edition, p. 352.
`"A System for Rapid DNA Sequencing with Fluorescent
`Chain-Terminating Dideoxynucleotides", J. Prober et al.,
`Science, vol. 238, Oct. 16, 1987, pp. 336-341.
`"A New Procedure for Determining Thymine Residues in
`DNA Sequencing. Photoinduced Cleavage of DNA Frag(cid:173)
`ments in the Presence of Spermine", I. Saito et al., Nu(cid:173)
`cleic Acids Research, vol. 12/No. 6 (1984) pp.
`2879-2885.
`
`(List continued on next page.)
`
`Primary Examiner-Mindy B. Fleisher
`Attorney, Agent, or Firm-William H. May; P. R.
`Harder; Janis C. Henry
`ABSTRACT
`[57]
`A method is provided for determining the sequence of
`nucleotides on a single strand DNA molecule. The
`single strand DNA molecule is attached to a leader
`oligonucleotide and its complementary strand to a solid
`state support. Fluorescently-labeled 3' -blocked nucleo(cid:173)
`tide triphosphates, with each of the bases A, G, C, T
`having a different fluorescent label, are mixed with the
`bound DNA molecule in the presence of DNA poly(cid:173)
`merase. The DNA polymerase causes selective addition
`of only the complementary labeled NTP, thus identify(cid:173)
`ing the next unpaired base in the unknown DNA strand.
`The 3'-blocking group is then removed, setting the
`system up for the next NTP addition and so on. The
`sequence is repeated until no more fluorcscently-labeled
`NTPs can be detected as being added by the polymer(cid:173)
`ase.
`
`10 Claims, 2 Drawing Sheets
`
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`
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`
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`
`Illumina Ex. 1042
`IPR Petition - USP 10,435,742
`
`Page 1
`
`

`

`5,302,509
`Page 2
`
`OTHER PUBLICATIONS
`"Optimized Conditions for Solid-Phase Sequencing: Si(cid:173)
`multaneous Chemical Cleavage of a Series of Long DNA
`Fragments Immobilized on CCS Anion-Exchange Pa(cid:173)
`per'', A. Rosenthal et al, Gene 42 (1986) pp. 1-9.
`"New Sequences to Take on the Genome", L. Smith,
`Research News (1987) pp. 271-272.
`"Selective Inhibition of DNA Chain Elongation Catalyzed
`by DNA Polymerase", A. K.rayevsky et al., Nucleosides
`& Nucleotides, 7 (5&6) pp. 613-617 (1988).
`"Fluorescence Detection in Automated DNA Sequence
`
`Analysis", L. Smith et al., Nature, vol. 321, Jun. 1986,
`pp. 674-679.
`"Direct Solid Phase Sequencing of Genomic and Plasmid
`DNA Using Magnetic Beads as Solid Supporf', T. Hult(cid:173)
`man et al., Nucleic Acids Research, vol. 17/No. 13
`(1989) pp. 4937-4946.
`"CIV. Total Synthesis of the Structural Gene for an Ala(cid:173)
`nine Transfer Ribonucleic Acid from Yeast, Chemical
`Synthesis of an lcosadeoxynucleotide Co"esponding to the
`Nucleotide Sequence 21 to 4(!', H. Weber et al, J. Mol.
`Biol. (1972) 72, pp. 219-249.
`
`Page 2
`
`

`

`U.S. Patent
`US. Patent
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`Apr. 12, 1994
`Apr. 12, 1994
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`U.S. Patent
`U.S. Patent
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`Apr. 12, 1994
`Apr. 12, 1994
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`

`

`1
`
`METHOD FOR SEQUENCING
`POLYNUCLEOTIDES
`
`5,302,509
`
`This application is a continuation of U.S. application 5
`Ser. No. 07/393,586 filed Aug. 14, 1989 now aban(cid:173)
`doned.
`The present invention is directed to a method for
`sequencing DNA molecules.
`
`10
`
`25
`
`BACKGROUND OF THE INVENTION
`The present invention provides a method for deter(cid:173)
`mining the nucleotide sequence of DNA molecules
`(referred to herein as the nucleotide base sequence or
`simply the base sequence). Several methods are known 15
`for sequencing DNA molecules such as methods of F.
`Sanger, S. Nicklen, A. R. Coulson, Proc. Natl. Acad. Sci.
`U.S.A., 74, 5463 (1977), and A. M. Maxam and W.
`Gilbert, Proc. Natl. Acad. ScL U.S.A., 74, 560 (1977).
`These known methods use various means for producing 20
`labeled fragments of DNA, each of which terminates
`with a known base (A, G, C or T). These fragments are
`then separated by length, typically by an electropho(cid:173)
`retic gel, utilizing a different gel strip for each type of
`terminal base. The DNA sequence is then read from the
`gel strips. As a variation, instead of using the same label
`for each fragment (such as a fluorescent dye or radioac(cid:173)
`tive label) J. M. Prober, et al., Science, 238, 336-341,
`Oct. 1987, and C. Connell et al., BioTechniques, Vol. 5, 30
`No. 4, 342-348 (1987), use a different dye to label each
`of the different base termination fragments so there is a
`different dye associated with A, G, C and T termina(cid:173)
`tion. This modification allows a single gel to be used,
`however, it also introduces new problems due to the 35
`effect of the different dyes on fragment mobility.
`A limitation of the prior methods is that they are
`apparently limited by the rate at which the fragments
`may be separated and are also limited by the number of
`bases that can be sequenced in a given run by the resolu- 40
`tion obtainable on the gel. The separation rate is inher(cid:173)
`ently limited, for example, by thermal distortion of the
`gel caused by electrical heating, and thus the identifica(cid:173)
`tion can only be obtained as often on average as about a
`few bases per minute. Also the resolution on the gel is a 45
`maximum of about 1,(X)() bases, with improvement in
`this resolution not being likely because of band com(cid:173)
`pression effects, and because there are interactions be(cid:173)
`tween the DNA strands which dominate over the
`length effect of very long strands, thus confusing the 50
`signal for long fragments.
`The present invention provides an improvement over
`these prior art methods.
`It is thus an object of the present invention to provide
`a method of DNA identification in which the rate limit- 55
`ing step is essentially the rate of a polymerase reaction,
`which is usually on the order of at least 60 bases per
`second, or limited by the rate in which the reagents can
`be delivered to the reaction site, whichever is slower.
`It is another object of the present invention to pro- 60
`vide a method of DNA sequencing in which the accu(cid:173)
`racy does not depend upon the length of the DNA
`molecule to be sequenced but, rather on the signal-to(cid:173)
`noise ratio of the detection means, which is very low
`using optical detection methods. Such high sensitivity 65
`detection means provide the advantage that only very
`small quantities of DNA are necessary, typically, less
`than a million molecules.
`
`2
`It is yet another object of the present invention to
`provide a DNA sequencing which is unambiguous even
`in short sequences of identical bases, which are difficult
`to distinguish by prior art methods.
`Another object of the present invention is to provide
`a novel method for DNA sequencing in which the rea(cid:173)
`gents for detection comprises a single mixture of bases,
`and does not require four separate preparations (one for
`each base) as required by methods of the prior art.
`These and other objects of the present invention will
`be apparent from the following description, the ap(cid:173)
`pended claims and from practice of the invention.
`
`SUMMARY OF THE INVENTION
`The present invention provides a method for deter(cid:173)
`mining the nucleotide sequence of a single strand DNA
`molecule comprising the steps of:
`(a) providing a set of identical single strand DNA
`molecules (ssDNA) comprising at the 3' end a
`leader sequence, the leader sequence comprising a
`region recognizable by a DNA polymerase for
`initiation of replication;
`(b) providing an oligonucleotide complementary to at
`least a portion of the leader sequence, and capable
`of forming a stable double stranded DNA hybrid
`therewith;
`(c) covalently attaching the 3' end of the leader se(cid:173)
`quence, the 5' end of the ssDNA or an end of the
`oligonucleotide to a solid support;
`(d) forming a stable double strand DNA hybrid
`bound to the solid support, the hybrid comprising
`the oligonucleotide and the single stranded DNA
`molecule with the leader sequence and the bound
`hybrid acting as a primer for DNA polymerase
`replication;
`(e) exposing the hybrid bound to the solid support to
`a DNA polymerase in the presence of fluorescent(cid:173)
`ly-labeled 3'-blocked derivatives of the four nucle(cid:173)
`otide 5' -triphosphates of 2' -deoxyadenosine, 2' -
`deoxyguanosine, 2'-deoxycytidine and 2'-deoxy(cid:173)
`thymidine, where each of the four nucleotide 5'-tri(cid:173)
`phosphate (NTPs) derivatives is labeled with a
`fluorescent label distinguishable by fluorescent
`detection means from the other three labels on the
`other three nucleotide 5' -triphosphate derivatives,
`under conditions whereby the polymerase will add
`the appropriate complementary nucleotide 5'-tri(cid:173)
`phosphate derivative to the oligonucleotide;
`(t) separating any unused NTP derivatives from the
`solid supported DNA hybrid and the support;
`(g) identifying the labeled NTP derivative added to
`the double stranded DNA by optical detection
`means;
`thereby identifying its complementary
`deoxynucleotide present in the single stranded
`DNA molecule;
`(h) removing the fluorescent label and 3' blocking
`group from the labeled NTP derivative of step (g)
`to expose the normal OH group in the 3'-position;
`(i) separating the freed blocking group and label
`(which may be associated with the blocking group)
`from the solid supported double stranded DNA
`hybrid;
`(j) repeating steps (e) through (i) through a plurality
`of cycles until labeled NTPs can no longer be
`added to the oligonucleotide; whereby the result of
`each cycle identifies the next deoxynucleotide in
`sequence in the single stranded DNA molecule.
`
`Page 5
`
`

`

`5,302,509
`
`o-
`
`0
`
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`
`\_ /
`
`X " DYE
`
`wherein:
`X is -0-A- and
`A is a functional group which is removable to expose
`the 3' -hydroxyl group.
`For example, A may be
`
`0
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`II
`-CCH2CH2C-NH-C:=C (Dye).
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`Another starting material required for the present
`BRIEF DESCRIPTION OF THE ORA WINGS
`invention is a mixture of four fluorescently-labeled (or
`In the accompanying FIG. 1 there is schematically
`other optically labeled such as optical absorption dyes,
`or chelated ions) 3'-blocked, NTPs (nucleotide triphos-
`shown a double stranded DNA hybrid bound to a solid
`5 phates). The preferred embodiment has the fluorescent
`support utilized in accordance with the present inven(cid:173)
`tion.
`label as part of the 3' -blocking group. Each of the NTPs
`In the accompanying FIG. 2 there is illustrated a
`will be labeled by a different label (i.e., each of the A, G,
`photolytic removal of a 3'-blocking group from a 3'(cid:173)
`C and T NTPs will have different labels on them) so as
`blocked nucleotide in accordance with the present in(cid:173)
`to be distinguishable by fluorescent spectroscopy or by
`vention.
`10 other optical means. Such labels are known in the art
`and are disclosed for example in Prober, et al., Science,
`DESCRIPTION OF THE PREFERRED
`vol. 238, pp. 336-341 (1987) and Connell et al., BioTech(cid:173)
`EMBODIMENTS
`niques, Vol. 5, No. 4, 342-384 (1987); Ansorge, et al.,
`The purpose of the present invention is to determine
`Nucleic Acids Research, vol. 15 (11) 4593-4602 (1987)
`the sequence of a set of identical single stranded DNA 15 and by Smith, et al., Nature:321, 674 (Jun. 12, 1986).
`molecules, therefore it will be assumed that such strands
`Each of the NTPs has a 3'-blocking group, so as to
`prevent the polymerase from continuing to replicate
`are initially provided. These strands, hereinafter called
`ssDNA, are adapted to be used in accordance with the
`once one base has been added. This is preferably accom-
`present invention. Various ways to adapt the ssDNA
`plished by having the dye attached through a cova-
`for attachment with a complementary oligonucleotide 20 Iently linking group to the 3'-position so that the dye
`to a solid support will be apparent to those of ordinary
`moiety and the 3'-blocking group are contained in the
`skill in the art. Several methods are described herein as
`same substituent. Examples are shown below.
`preferred embodiments. One method is to modify the
`ssDNA with a known leader. The purpose of the modi(cid:173)
`fication is to attach to the 3' end of the ssDNA a known 25
`leader sequence which (when hybridized for form a
`duplex) is recognizable by the polymerase to be utilized
`for the initiation of replication unless the provided
`ssDNA already has a known leader sequence. The 3'(cid:173)
`end of the leader may also provide a handle which may 30
`be attached to a solid support. The ssDNA may be
`modified at least in any of the following ways.
`One such method is to first amplify the quantity of the
`ssDNA by polymerase chain reaction techniques (PCR,
`reference, R. Saiki et al., Science, Vol. 239, 487-491, 35
`January 1988). Prior to amplification, if necessary, a
`known single stranded sequence comprising the single
`stranded sequence of a sticky end may be added as a
`short, temporary leader to the ssDNA. Methods of
`joining the S' end of one oligonucleotide to the 3' end of 40
`another DNA molecule are known in the art and can be
`routinely performed.
`Amplification by PCR creates many such DNA mol(cid:173)
`ecules with a short leader. An oligo complementary to
`the short leader may then be added so that there is a 45
`This may be prepared by treating any of the reactive
`short section of double stranded DNA at the leader, as
`dyes, such as reactive fluorescent dyes functionalized
`shown in FIG. 1.
`An alternative approach is to attach a double
`with halo groups, with N-trifluoroacetyl propargyla-
`mine under conditions described by Prober et al., Sci-
`stranded oligo with a sticky end to the solid support.
`This oligo will have the complementary sequence of the 50 ence, 238: 336-341, Oct. 1987, then deacylating. This
`same restriction site used to create the ssDNA. Then
`amino-dye may then be coupled to a 3'-0-succinyl pro-
`the two sticky ends will be ligated to form a double
`tected nucleotide to produce a 3'-0-protected nucleo-
`stranded DNA molecule attached to a solid support as
`tide wherein the protecting group is
`shown in the FIG. 1.
`Alternatively, a single strand leader may be ligated to 55
`the end of the unknown ssDNA strand. The oligo con(cid:173)
`taining a sequence complementary to the leader (or
`portion thereof) may be bound through its S' end to the
`substrate. Then the ssDNA and the associated leader
`will be bound to the solid support by hybridization to 60
`the bound oligonucleotide to result in the identical situ(cid:173)
`ation shown in FIG. 1.
`Alternatively instead of sticky ends as described
`above, blunt end ligation may be utilized.
`Initially, the double stranded portion of the bound 65
`molecule in FIG. 1 will be a primer for a suitable DNA
`polymerase, preferably Tag polymerase, which is oper(cid:173)
`able at high temperature.
`
`which is removable under conditions similar to that of
`the 0-succinyl protecting group commonly used in
`solid phase nucleotide synthesis. Alternatively, an acid
`anhydride, to which the dye is attached, may be directly
`condensed with the 3'-0H group. Thus to initiate the
`sequencing, the bound double strand DNA molecule
`shown in FIG. 1 is exposed to the DNA polymerase and
`a mixture of the four fluorescently-labeled 3' -blocked
`NTPs. The polymerase will then add one of the four
`NTPs to the growing oligonucleotide chain, whichever
`
`Page 6
`
`

`

`5,302,509
`
`3PVSE
`
`5
`6
`This removal may be accomplished by chemical means
`NTP is complementary to the next unpaired base in the
`ssDNA. This step is rapid since the average reaction
`or photochemical means. The chemical means of re-
`moving the 3'-blocker will of course depend upon the
`rate of adding a base to an oligonucleotide with a poly-
`nature of the 3'blocking groups, many of which are
`merase is in the range of at least 60 bases per second.
`Since only one base is being added this can be accom- S known in the art as shown for example in Chapter 1,
`plished in less than a second.
`Organic Chemistry of Nucleic Acids, Part B, Eds. N. K.
`The next step is to separate the unused NTP's from
`Kochetkov and E. I. Budovskii, Plenum Press, 1972 and
`H. Weber and H. G. Khorana, J. Mo/. Biol., 72, 219-249
`the vicinity of the support bound DNA by washing.
`Since it is possible for the free NTPs to bind to the
`(1972). Methods of their removal are therefore also
`ssDNA, the wash should take place at a temperature so 10 known.
`that the free NTPs do not bind to the ssDNA, but not
`Preferably the fluorescent label and 3'-blocker may
`high enough to dehybridize the double stranded DNA.
`be removed quickly by photolysis, as shown in FIG. 2.
`In order not to deactivate the polymerase at such a
`In the photolysis reaction, instead of the absorbed light
`energy being re-emitted as fluorescent light, it is occa-
`temperature, it is preferred that a high temperature
`polymerase be utilized such as the aforementioned taq 15 sionally conveyed to the 3• position by means of an
`alternating single-double bonds hydrocarbon chain.
`polymerase. Since the double stranded DNA is in the
`environment of a solid support, it is also required that
`the solid support surface not attract the NTPs, so Tef-
`The excitation energy can then catalyse (or enable) the
`Ion or similarly non-adhering lining for the solid sup-
`hydrolysis of the acyl ~~up,. as show~, Even: ~f!er
`port should be utilized. The wash which is used to wash 20 cleavag~ of~he d~e la~l, 1t 1s still present m the VIcm~ty
`the free NTPs from the support bound double stranded
`for optical 1d~nt~ficat1on. The dyes are chosen with
`DNA may be any convenient wash such as buffered
`fluor~nt emiss1?n spectra th~t are as well separated
`saline, or polymerase buffer without the NTPs.
`as poss1bl~ _but which can be activated J?re~erably ~y ~he
`Washing is also a rapid step since it is contemplated ~e exc1tmg_sou!ce. ~us the onl~ s1gru_ficant hmita-
`that one would only be using small quantities of DNA 25 t1on to detec!1on 1s the s1gnal-to-n~1se ratio due to _the
`concentrated in a small area. The washing should only
`Rama scattenng of the other matenals present, part1cu-
`take around a few seconds or Jess.
`larly water. By using activation wavelengths which
`Since only one of the four types of NTPs will be
`minimize Raman scattering from water the signal-to-
`added to the oligonucleotide, it may be read by its fluo-
`noise ratio will be improved.
`The solid-supported ssDNA will be scanned for the
`rescent label using a suitable fluorescent detection 30
`means. According to the preferred embodiment of the
`optical labels preferably using a optical fiber. The end
`of the fiber may simply be brought close to the surface
`present invention this may be done by making the solid
`support for the bound DNA the end of an optical fiber.
`of the support for detection, or the end of the fiber may
`By transmitting the radiation of appropriate exciting
`itself be the solid support. An alternative means is to
`wavelength through the fiber, the labelled DNA at the 35 attach the DNA to the sides of the core of an optical
`fiber (by removing the cladding from a selected area). If
`end of the fiber will fluoresce and emit the light of
`appropriate fluorescent frequency. The emitted fluores-
`attached to the sides of an optical fiber, the DNA is
`cent light will be partially transmitted back into the
`illuminated by evanescent lighting surrounding the ti-
`optical fiber in the reverse direction to the exciting
`ber, and only light emission in this region can couple
`~-
`~~~~~
`This back propagating light can be separated spec-
`Concentration of the dye in a small area will also
`trally by such means as an etched diffraction grating on
`improve detection by increasing the ratio of fluorescent
`the fiber. The returned light spectrum identifies the
`material to background Rama scattering solution. The
`particularly bound NTP. It will be within the skill of
`optical fiber improves the signal-to-noise ratio com-
`those of ordinary skill in the art to adjust the concentra- 45 pared to methods of the prior art since the prior art
`tion of the bound DNAs such that there will be a suffi-
`illuminates comparatively large volumes. Also the use
`of an optical fiber to bring the light to the reaction area
`cient number of fluorescent molecules present for opti-
`cal detection by this means.
`and to carry the fluorescent output away is simple and
`It is preferred that the fluorescent dye marker and the
`can be used to separate the return light into spectral
`3' -blocking group be present in the same substituent, for 50 bands which can be then detected by small solid state(cid:173)
`example as shown below.
`like detection means such as PIN photodiodes. Spectral
`separation may be accomplished for example., by peri(cid:173)
`odic diffraction grading etched into the fiber or by
`using a cladding with a higher dispersion than the core
`ss so that different wavelengths will or will not satisfy the
`critical angle condition. Wavelengths that do not satisfy
`the critical angle condition will escape into the cladding
`where they can be detected. Utilizing solid state compo(cid:173)
`nents, the whole sequencing cell, including the optical
`60 detection means may be provided in a planar integrated
`optical system, which can thus be produced by photo(cid:173)
`lithographic means, as disclosed, for example in Planar
`Optical Wayeguides and Fibres, H. G. Unger, Clarendon
`Press, Oxford (1977). Planar optics also allows many
`65 sequencing cells to be produced on the same substrate
`and illuminated by the same light source. Signal-to(cid:173)
`noise ratios can be further improved by using time re(cid:173)
`solved fluorescence (TRF), with relatively long-lived
`
`The identification of the bound NTP identifies its
`complementary base as the first unpaired base in the
`unknown sequence of the ssDNA.
`The next step is to remove the 3 '-block and associated
`fluorescent label from the bound NTP to prepare for
`the addition of the next NTP onto the oligonucleotide.
`
`0
`
`o=(,
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`\DYE
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`5,302,509
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`15
`
`7
`fluorescent species (e.g., chelated rare earth ions). By
`this method the detection occurs after the Raman scat(cid:173)
`tering has subsided, but the fluorescent species are still
`in the emission mode, as disclosed in I. Hemmila, et al.,
`Anal. Biochem. 137, 335-343 (1984), for example.
`Once the fluorescent label and 3'-blocker (which are
`preferably one and the same) are removed from the
`labeled bound NTP, they are separated from the bound
`DNA by washing, using, for example, washing reagents
`described above.
`Then the sequence beginning with exposure to the
`polymerase and the four labeled 3'-block NTPs is re(cid:173)
`peated, with each cycle adding another base to the
`growing oligonucleotide, thereby identifying the next
`base in the unknown ssDNA sequence and so on. The
`identification is completed when a cycle fails to show
`that a label is present after the polymerase reaction and
`washing step.
`According to a preferred embodiment of the present
`invention, the double stranded DNA is attached onto 20
`the end of an optical fiber, and the end of the fiber may
`be dipped into each of the reagents in tum. By moving
`or vibrating the fiber, there will be rapid mixing of the
`boundary layer in the bulk reagent.
`Simple flow systems may be used to bring the rea- 25
`gents to the active part of the fiber, such as capillary
`electrophoresis or hydrostatic pressure, connected to
`the appropriate reagents.
`It is expected that all of the bound DNA molecules
`will start at the same position on the ssDNA and add the 30
`same type of NTP during each cycle of the process.
`Due to imperfect mixing or the dynamics of a densely
`populated surface the rate of adding NTPs to each of
`the solid state-bound DNAs may not be identical.
`Therefore some of the strands may fall behind in the 35
`sequence of adding an NTP. For example, if a 3'(cid:173)
`blocker remains after the block removal step for a par(cid:173)
`ticular DNA unit, that blocker may not be removed
`until the next cycle and therefore that particular strand
`will fall behind by addition of one NTP unit. The symp- 40
`toms of the loss of synchronization of adding an NTP
`will be the appearance in the fluorescent spectra of dyes
`other than the majority dye being detected for the NTP
`which was added. As long as the interfering fluorescent
`spectra do not interfere substantially with detection of 45
`the majority dye being added in the cycle of interest,
`this need not be a concern. For sufficiently long
`ssDNA, the accumulation of errors may require re-syn(cid:173)
`chronization, forcing the lagging DNA to catch up with
`the current DNA. This resynchronization can be 50
`achieved by changing the reaction mixture of the NTPs,
`and having two identical reaction cells, one running a
`few bases ahead of the other. For example if the se(cid:173)
`quence in the leading cell is ACGTTC, and the trailing
`cell is one base behind (i.e., ACGTT); then instead of 55
`adding the usual four blocked NTPs to the trailing cell,
`blocked C, A, G may be added with unblocked T.
`Those DNA units that are behind in the trailing cell
`(e.g., ACGT or ACG) add the unblocked T (or TT),
`followed by a blocked C, while the majority will just 60
`add the blocked C. The leading cell is necessary so that
`the next base for the majority of the trailing cell is
`known, and so an unblocked NTP can be added without
`interfering with the process. The roles of leading and
`trailing cells can be switched so that one can act as the 65
`predictor for the other, and vice versa.
`Referring to FIG. 1 there is shown a solid substrate
`10 covalently linked through a linker 11. In the FIG-
`
`8
`URE the linker 11 is attached to the 5' end of the oligo(cid:173)
`nucleotide 12, however it may also be attached to the 3'
`end of the ssDNA 13. The shaded area 14 is the DNA
`polymerase, which is primed by the oligo bound to the
`5 ssDNA.
`Having described the preferred embodiments of the
`present invention various modifications will be evident
`to those of ordinary skill in the art from the above de(cid:173)
`scription as well as from practice of the invention.
`10 These modifications are intended to be within the scope
`of the invention and the invention is not intended to be
`limited in any way except by the scope of the following
`claims.
`What is claimed is:
`1. A method for determining the nucleotide sequence
`of identical single strand DNA molecules comprising
`the steps of:
`(a) providing said single strand DNA molecules at
`their 3' end with a known leader sequence, said
`leader sequence forming a double stranded DNA
`hybrid with an oligonucleotide having a sequence
`complementary to said leader sequence;
`(b) providing to said leader sequence said oligonucle(cid:173)
`otide having a sequence complementary;
`(c) covalently attaching either the 3' end of said
`leader or the 5' end of said oligonucleotide to a
`solid support;
`(d) form4Ig a stable double stranded DNA hybrid,
`said hybrid comprising said oligonucleotide and
`said leader;
`(e) exposing said hybrid to a DNA polymerase in the
`presence of optically-labeled derivatives of four
`nucleotide 5'-triphosphates of 2'-deoxyadenosine,
`2'-deoxyguanosine, 2'-deoxycytidine and 2'-deoxy(cid:173)
`thymidine, where said optically-labeled derivatives
`comprise a blocking group at the 3' portion thereof,
`said blocking group comprising an optical label
`capable of being removed to expose the 3' portion
`thereof, where each of said four derivatives is la(cid:173)
`beled with an optical label distinguishable by an
`optical detection means capable of detecting said
`optical label from the other three labels on the
`other three of said derivatives under conditions
`whereby said polymerase will add the complemen(cid:173)
`tary optically-labeled 3' -blocked nucleotide 5' -tri(cid:173)
`phosphate to said oligonucleotide;
`(0 washing any unused derivatives from said double
`stranded DNA hybrid;
`(g) detecting the labeled derivative incorporated onto
`the double stranded DNA hybrid by said optical
`detection means thereby identifying the comple(cid:173)
`ment of said optically-labeled 3'-blocked nucleo(cid:173)
`tide 5' -triphosphate added to said oligonucleotide
`present in said single stranded DNA molecules;
`(h) removing, by chemical means or photochemical
`means, the optically-labeled 3'-blocking group
`from the derivative incorporated in step (g) to
`expose the OH group in the 3' position; of the nu(cid:173)
`cleotide 5' -triphosphate derivative incorporated
`into the double stranded DNA hybrid
`(i) separating the removed optically-labeled blocking
`group from said double stranded DNA hybrid;
`G) repeating steps (e) through (i) through a plurality
`of cycles until labeled nucleotide 5'-triphosphate
`derivatives can no longer be added to said oligonu(cid:173)
`cleotide, whereby the result of each cycle identifies
`the next deoxynucleotide in sequence in said single
`stranded DNA molecules.
`
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`5,302,509
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`9
`2. A method according to claim 1 wherein said poly(cid:173)
`merase comprises Taq polymerase.
`3. A method according to claim 1 wherein said opti(cid:173)
`cal labels comprise fluorescent derivatives which fluo(cid:173)
`resce upon excitation thereof by irradiation with an
`appropriate radiation wavelength.
`4. A method according to claim 3 wherein said opti(cid:173)
`cal labels are a part of said 3'-blocking group.
`5. A method according to claim 4 wherein said 3'- 10
`blocking group is removed from said nucleotide tri(cid:173)
`phosphates by photochemical activation.
`6. A method according to claim 1 wherein the 3' end
`of said leader is attached to said single stranded DNA
`molecule by polymerase chain reaction amplification.
`7. A method according to claim 1 wherein the 3' end
`of said leader is attached to said single stranded DNA
`molecule by blunt end ligation.
`
`10
`8. A method according to claim 1 wherein the 3' end
`of said leader is attached to said single stranded DNA
`molecule by stick