PCT
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`WO 91/06678
`(11) International Publication Number:
`(51) International Patent Oassification 5 :
`C12Q 1/68, C12P 19/34
`// C07H 21/00, 21/04, GOIN 35/08
`
`Al
`
`( 43) International Publication Date:
`
`16 May 1991 (16.05.91)
`
`(21) International Application Number:
`
`PCT/US90/06178
`
`(22) International Filing Date:
`
`26 October 1990 (26.10.90)
`
`(74)Agents: CHEN, John, Y.; SRI International, 333 Raven(cid:173)
`swood Avenue, Menlo Park, CA 94025-3493 (US) et al.
`
`(30) Priority data:
`427,321
`
`26 October 1989 (26.10.89)
`
`us
`
`(71)Applicant: SRI INTERNATIONAL [US/US]; 333 Raven(cid:173)
`swood Avenue, Menlo Park, CA 94025-3493 (US).
`(71 X72) Applicant and Inventor: TSIEN, Roger, Y. [US/US];
`8535 Nottingham Place, La Jolla, CA 92037-2125 (US).
`(72) Inventors: ROSS, Pepi ; 745 Contra Costa Avenue, Berke(cid:173)
`ley, CA 94707 (US). FAHNESTOCK, Margaret ; 2724
`Gamble Court, Hayward, CA 94542 (US). JOHNSTON,
`Allan, J. ; 909 North California Avenue, Palo Alto, CA
`94303 (US).
`
`(81) Designated States: AT (European patent), BE (European
`patent), CA, CH (European patent), DE (European pa(cid:173)
`tent), DK (European patent), ES (European patent), FR
`(European patent), GB (European patent), GR (Euro(cid:173)
`pean patent), IT (European patent), JP, LU (European
`patent), NL (European patent), SE (European patent).
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(54) Title: DNA SEQUENCING
`
`(57) Abstract
`The present invention relates to an instrument and a method to determine the nucleotide sequence in a DNA molecule
`without the use of a gel electrophoresis step. The method employs an unknown primed single stranded DNA sequence which is
`immobilized or entrapped within a chamber with a polymerase so that the sequentially formed cDNA can be monitored at each
`addition of a blocked nucleotide by measurement of the presence of an innocuous marker on specified deoxyribonucleotides. The
`invention also relates to a method of determining the unknown DNA nucleotide sequence using blocked deoxynucleotides. The
`blocked dNTP has an innocuous marker so that its identity can be easily determined. The present instrument and method provide
`a rapid accurate determination of a DNA nucleotide sequence without the use of gel ele<:trophoresis.
`
`Page i
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`Illumina Ex. 1031
`IPR Petition - USP 10,435,742
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`

`

`DESIGNATIONS OF "DE"
`
`Until further notice, any designation of "DE" in any international application
`whose international filing date is prior to October 3, 1990, shall have effect in the
`territory of the Federal Republic of Germany with the exception of the territory of the
`former German Democratic Republic.
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCf on the front pages of pamphlets publishing international
`applications under the PCT.
`
`AT
`AU
`BB
`BE
`BF
`BG
`BJ
`BR
`CA
`CF
`CG
`CH
`Cl
`CM
`DE
`DK
`
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Bra.,.il
`Canada
`Central African Republic
`Congo
`Swit:t.erland
`Cote d'Ivoire
`Cameroon
`Germany
`Denmark
`
`ES
`Fl
`FR
`GA
`GB
`GR
`HU
`IT
`JP
`KP
`
`KR
`LI
`L.K
`LU
`MC
`
`Spain
`Finland
`France
`Gabon
`Uniled Kingdom
`Greece
`Hungary
`Italy
`Japan
`Democratic People's Rcpuhlic
`of Korea
`Republic of Korea
`Liechtenstein
`Sri Lanka
`Luxembourg
`Monaco
`
`MG
`ML
`MR
`MW
`NL.
`NO
`PL
`RO
`SD
`SE
`SN
`SU
`TD
`TG
`us
`
`Madagascar
`Mali
`Mauritania
`Malawi
`Netherlands
`Norway
`Poland
`Romania
`Sudan
`Sweden
`&mcgal
`Soviet Union
`Chad
`Togo
`United States of America
`
`t
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`35
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`DNA SEQUENCING
`
`Background Of The Invention
`
`Field of the Invention
`This invention relates to DNA sequencing. More
`particularly, it relates to methods and apparatus for
`determining the sequence of deoxyribonucleotides within
`DNA molecules.
`
`Description of Background Art
`DNA sequencing is an important tool. A current
`goal of the biological community in general is the
`determination of the complete structure of the DNA of a
`number o_f organisms, including man. This information will
`aid in the understanding, diagnosis, prevention and
`treatment of disease.
`Current DNA sequencing methods employ either
`chemical or enzymatic procedures to produce labeled
`In the chemical method,
`fragments of DNA molecules.
`reactions are performed that specifically modify certain
`of the nucleotide bases present in the end-labeled DNA.
`These reactions are carried out only partially to
`completion so that only a portion of the bases present in
`the molecules are reacted. These modified bases are then
`treated with piperidine, to cleave the DNA chains at the
`modified bases producing four sets of nested fragments.
`These fragments are then separated from one another
`according to size by electrophoresis in polyacrylamide
`gels: The fragments can then be visualized in the gels by
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`means of radioactive labels. The position of the
`fragments in the gel indicates the identity of the last
`nucleotide in each fragment so that on the gel a "ladder"
`of fragments, with each step identified, is assembled to
`5 provide the overall sequence.
`In the enzymatic method, the DNA to be sequenced
`is enzymatically copied by the Klenow fragment of DNA
`polymerase I or by a similar polymerase enzyme such as Taq
`polymerase or Sequenase™. The enzymatic copying is carried
`In each of the four reactions a low
`10 out in quadruplicate.
`concentration of a chain terminating dideoxynucleotide is
`present, a different dideoxynucleotide being present in
`each of the four reactions (ddATP, ddCTP, ddGTP and
`ddTTP). Whenever a dideoxynucleotide is incorporated, the
`15 polymerase reaction is terminated, again producing sets of
`nested fragments. Again, the nested fragments have to be
`separated from one another by electrophoresis to determine
`the sequence.
`Recently, new advances in sequencing technology
`.20 have introduced automated methods. Applied Biosystems has
`developed an instrument based on the use of fluorescent
`labels and a laser-and computer-based detection system
`(Smith et al., 1986; Smith, 1987). An automated system
`developed by E.E. du Pont de Nemours & Company, Inc.
`(Prober et al., 1987) is similar to the Applied Biosystems
`instrument but uses fluorescently labeled ddNTPs to
`terminate the reaction instead of fluorescent primers.
`Hitachi (Japan) and EMBL (West Germany) have developed
`similar systems (Ansorge et al., 1986). Other approaches
`involve multiplexing technology (Church and
`Kieffer-Higgins, 1988), detection of radioactively labeled
`DNA fragments by sensitive Beta-detectors (EG&G),
`automated gel readers (BioRad), and automated liquid
`handlers (Beckman Instruments; Seiko; Goodenow, University
`35 of California, Berkeley).
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`The need to rely on electrophoresis and a
`separation according to size as part of the analytical
`scheme is a severe limitation. The gel electrophoresis is
`a time-consuming step and requires very highly trained
`5 skilled personnel to carry it out correctly. The present
`invention provides methods and apparatus for sequencing
`DNA which do not require electrophoresis or similar
`separation according to size as part of their methodology.
`
`20
`
`10 References of Interest
`The following articles and patents relate to the
`general field of DNA sequencing and are provided as a
`general summary of the background art. From time to time
`reference will be made to these items for their teaching
`15 of synthetic methods, coupling and detection
`In these cases, they will
`methodologies, and the like.
`generally be referred to by author and year.
`W.B. Ansorge, et al., (1987) Nucleic Acid
`Research, 15:4593-4602.
`W.B. Ansorge, et al., (1986) Journal of
`Biochemical and Biophysical Methods, 13:325-323.
`J. T, Arndt-Jovin, et al., (1975) European
`Journal of Biochemistry, 54:411-413.
`H. Bunemann, et al. (1982) Nucleic Acids
`25 Research, 10:7163-7180.,
`L.D. Cama, et al., (1978) Journal of the
`American Chemical Society, 100:8006.
`G. M, Church, et al., (1988) Science
`240:1ss-1es.
`S.A. Chuvpilo, et al., (1984) "A Simple and
`FEBS 179:34-36.
`Rapid Method for Sequencing DNA,"
`L.F. Clerici, et al., (1979) Nucleic Acids
`Research, 6:247-258.
`L.A. Cohen, et al., (1966) Journal of Organic
`35 Chemistry, 31:2333.
`
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`B.A. Connolly, (1987) Nucleic Acids Research,
`15:3131-3139.
`C.G. Cruse, et al., (1978) Journal of Organic
`Chemistry, 43:3548-3553.
`P.T. Englund, et al., (1969) Journal of
`Biological Chemistry, 244:3038-3044.
`B.C. Froehler, et al., (1986) Nucleic Acids
`Research, 14:5399-5407.
`R. Gigg, et al., {1968) Journal of the Chemical
`10 Society, Cl4:1903-1911.
`P.T. Gilham, (1968) Biochemistry, 7:2809-2813.
`M.L. Goldberg, et al., (1979) Methods in
`Enzymology, 68:206-220.
`T. Goldkorn, et al., (1986) Nucleic Acids
`15 Research 14:9171-9191.
`T.W. Greene, (1981) Protective Groups in Organic
`Synthesis, John Wiley and Sons, Inc., New York, New York.
`E. Hansbury, et al., (1970) Biochemical &
`Biophysical Acta, 199:322-329.
`C. Hansen, et al., (1987) Analytical
`Biochemistry, 162:130-136.
`W.D. Henner, et al., (1983) Journal of
`Biological Chemistry; 258:151198-15205.
`J.A. Huberman, et al., (1970) Journal of
`25 Biological Chemistry, 245:5326-5334.
`Y. Kanaoka, (1977), Angewante Chemie
`International Edition English, 16:137-147.
`A. Kornberg,. (1974), DNA Synthesis, w. H.
`Freeman and Company, San Francisco.
`A.A. Kraevskii, et al., (1987) Molecular
`Bioloay, 21:25-29.
`A.A. Kraevsky, et al., (1987) Biophosphates and
`Their Analogues--Synthesis, Structure, Metabolism and
`Activity, K.S. Bruzik and W.J. Stec (Eds.), Elsevier,
`35 Amsterdam, pp. 379-390 (and references therein).
`
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`J.N. Kremsky, et al., (1987) Nucleic Acids
`Research, 15:2891-2909.
`T.V. Kutateladze, et al., (1987) Molecular
`Biology, 20:222-231.
`J.A. Langdale, et al., (1985) Gene 36:201-210.
`R.T. Letsinger, et al. (1964) Journal of Organic
`Chemistry, 29:2615-2618.
`J.K. Mackey, et al., (1971) Nature, 233:551-553.
`T. Maniatis, et al., (1982) Molecular Cloning, A
`10 Laboratory Handbook, Cold Spring Harbor Laboratory, Cold
`Spring Harbor, New York.
`A. M. Maxam, et al., (1980) Methods in
`Enzymology, 65:499-560.
`E. Ohtsuka, et al., (1978) Journal of the
`15 American Chemical Society, 100:8210-8213.
`A.V. Papchikhin, et al., (1985) Bioorganic
`Chemistry, 11:716-727.
`s. Pochet, et al., (1987), Tetrahedron,
`43:3481-3490.
`R. Polsky-Cynkin, et al., (1985) Clinical
`Chemistry, 31:1438-1443.
`J.M. Prober, et al., (1987) Science,
`238:336-341.
`C.B. R~ese, et al. (1968) Tetrahedron Letters,
`25 40:4273-4276.
`T.A. Rezovskaya, et al., (1977) Molecular
`Biology, 11:455-466.
`F. Sanger, et al., (1977) Proceedings of the
`National Academy of Science USA, 74:5463-5467.
`s.R. Sarfati, et al. (1987) Tetrahedron Letters,
`43:3491-3497.
`B. Seed, (1982) Nucleic Acids Research,
`10:1799-1810.
`A.J.H. Smith, (1980) Methods in Enzymology,
`35 65:560-580.
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`L.M. Smith, et al., (1986) Nature, 321:674-679.
`L.M. Smith, (1987) Science, 235:G89.
`E.M. Southern, (1975) Journal of Molecular
`Biology, 98:503-517.
`s. Tabor, et al., (1987) Proceedings of the
`National Academy of Sciences USA, 84:4767-4771.
`R.I. Zhdanov, et al., (1975) Synthesis,
`1975:222-245.
`
`5
`
`15
`
`10 Additional references of interest are:
`N. Dattagupta, U.S. Patent No. 4,670,380 issued
`June 2, 1987.
`W.J. Martin, European Patent Application No.
`0187699, published July 16, 1986.
`Japan_~okai Tokyo Kobo JP 58/87,452 (May 25,
`1983); Chern. Abs, Vol. 99, No. 172376n.
`R. Lewis, "Computerizing Gene Analyses" High
`Technology, December 1986, p. 4p ff.
`C. Connell, et al. "Automated DNA Sequence
`20 Analysis'', BioTechnigues, Vol. 5, No. 4, p. 342 ff.
`(1987).
`
`J.F.M. De Rooiz, et al., Journal of
`Chrornotography, Vol. 177, p. 380-384 (1987).
`
`25
`
`Statement of the Invention
`
`The present invention provides methods and
`apparatus for determining the sequence of
`30 deoxyribonucleotides in a DNA molecule. A key
`characteristic of this invention is that it determines the
`DNA sequence without recourse to electrophoresis or other
`size-based separation techniques.
`In one aspect, the present invention provides a
`35 method for determining the deoxyribonucleotide sequence of
`a single stranded DNA subject molecule. This method
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`involves synthesizing, in the presence of a multitude of
`identical copies of the subject DNA, the DNA molecule
`which is complementary to it. This synthesis is carried
`out using deoxyribonucleotide triphosphates (dNTP) in a
`5 stepwise serial manner so as to simultaneously build up
`numerous copies of the complementary molecule, dNTP by
`dNTP. As each dNTP is added to the growing complementary
`molecules, it is identified by way of an appropriate label
`(i.e., reporter group). By noting the identity of the
`10 bases present in this complementary molecule and using
`standard rules of DNA complementation, one can translate
`from the complementary molecule to the corresponding
`original subject molecule and thus obtain the
`deoxyribonucleotide sequence of the subject molecule.
`In an additional aspect, this invention provides
`apparatus for carrying out the above-described method.
`As will be seen in the Detailed Description of
`the Invention which follow~, this method and apparatus for
`carrying it out can take many different configurations. A
`20 key to all of them, however, is the fact that the DNA
`sequence is determined not by generating a series of
`nested fragments which must b~ separated according to size
`but rather by direct identification of the dNTPs as they
`are incorporated into the growing complementary DNA chain.
`This invention can be carried out in a single
`reaction zone with multiple differentiable reporters or in
`multiple reaction zones with a single reporter in each
`It can be carried out by detecting the incremental
`zone.
`signal chijnge after addition of reporters or by noting
`30 each added reporter separately. The various reporters can
`be measured in the reaction zones while attached to the
`growing molecule or they can be separated from the
`molecule and then measured.
`The invention can be practiced to create the
`35 growing complementary DNA chain without interruption or it
`can be practiced in stages wherein a portion of the
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`complementary chain is created and its sequence
`determined; this portion of the chain is then removed; a
`sequence corresponding to a region of the removed chain is
`separately synthesized and used to prime the template
`5 chain for subsequent chain growth. The latter method can
`be repeated as needed to grow out in portions the complete
`complementary chain.
`
`10
`
`Detailed Description of the invention
`
`Brief Description of the Drawings
`The invention will be further described with
`reference being made to the accompanying drawings in
`15 which:
`
`20
`
`Figures lA and lB are schematic diagrams of the
`process of this invention on a molecular level.
`Figure 2 is a schematic representation of one
`form of apparatus for practising the invention.
`In this
`embodiment the DNA growth takes place in a single reaction
`zone. This embodiment uses separate, distinguishable
`reporters associated with each of the four nucleotides
`incorporated into the growing molecule. The four
`qifferent reporters are measured after each addition to
`25 detect which base has just been added to that position of
`the complementary chain.
`Figure 3 is a schematic representation of
`another form of apparatus for practising the invention.
`This embodiment employs four reaction zones in which the
`30 molecular growth is carried out in quadruplicate.
`In each
`of the four zones, a different one of the four nucleotides
`is associated with a reporter (with the remaining three
`being unlabeled) so that the identity of the nucleotide
`incorporated at each stage can be determined.
`Figure 4 is a schematic representation of an
`adoption of the apparatus for practising the invention
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`particularly adapted for carrying out the invention to
`grow a series of portions of the complementary molecule as
`opposed to a single continuous complementary molecule.
`Figures 5 through 8 are pictorial
`representations of chemical reaction sequences which can
`be used to synthesize representative labeled nucleotide
`building blocks for use in the practice of this invention.
`
`5
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`10
`
`Organization of this Section
`This Detailed Description of the Invention is
`organized as follows:
`First, several terms are defined in a
`Nomenclature section.
`Second, a series of Representative Apparatus
`15 Configurations and Process Embodiments for carrying out
`the invention are described.
`Third, Materials and Reagents and Methods of Use
`employed in the process of the invention are set forth,
`including;
`Enzymes and Coupling Conditions,
`Blocking Groups and Methods for Incorporation,
`Deblocking Methods,
`Reporter Groups, their Incorporation and
`Detection, and
`Immobilization of Subject DNA.
`Thereafter, a series of nonlimiting EXAMPLES is
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`provided.
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`Nomenclature
`A number of related and generally conventional
`abbreviations and defined terms appear in this
`specification and claims. The four nucleotides are at
`times referred to in shorthand by way of their nucleoside
`bases, adenosine, cytidine, guanosine and thymidine, or
`"A", "C", "G" and "T''. Deoxynucleotide triphosphates
`"dNTPs" of these materials are abbreviated as dATP, dCTP,
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`dGTP and dTTP. When these materials are blocked in their
`3'-OH position they are shown as 3'blockedd.ATP,
`3'blockeddCTP, 3'blockeddGTP and 3'blockeddTTP.
`Similarly, when they are each tagged or labeled with a
`5 common reporter group 1 such as a single fluorescent group,
`they are represented as d.A'TP, dC'TP, dG'TP and dT'TP.
`When they are each tagged or labeled with different
`reporter groups, such as different fluorescent groups,
`they are represented as d.A'TP, dC''TP, dG'''TP and
`10 dT''' 'TP. As will be explained in more detail below, the
`fact that the indication of labeling appears associated
`with the "nucleoside base part'' of these abbreviations
`does not imply that this is the sole place where labeling
`can occur. Labeling could occur as well in other parts of
`the molecule.
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`Representative Apparatus Configurations and Process
`Embodiments
`In the specification and claims, reference is
`20 made to a "subject" DNA or "template" DNA to define the
`DNA for which the sequence is desired.
`In practice, this
`material is contained within a vector of known sequence.
`A primer, which is complementary to the known sequence of
`the vector is used to start the growth of the unknown
`25 complementary chain. Two embodiments of this process are
`illustrated on a molecular level in Figures lA and lB.
`In Figure lA, a solid support 1 is illustrated
`with a reactive group A attached to its surface via tether
`2. This attachment can be covalent, ionic or the like. A
`second reactive group.X, capable of bonding to group A,
`again via a covalent, ionic or the like bond, is attached
`to the 5' end of a DNA primer 4. This primer has a known
`DNA sequence. When coupled to the substrate via the A-X
`bond it forms immobilized primer 5. Primer 5 is then
`35 hybridized to template DNA strand 6 which is made up of an
`unknown region 7 inserted between regions 8 and 8'.
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`Regions 8 and 8' are located at the 5' and 3' ends of the
`unknown region and have known sequences. The 8' region's
`known sequence is complementary to the sequence of primer
`4 so that those regions hybridize to form immobilized
`template DNA 9. Therefore the individual dNTPs are
`serially added to form the DNA sequence complementary to
`11 and 12 represent
`the unknown region of the template.
`the first two such dNTPs incorporated into the growing
`molecule. These in turn provide the identity of their
`complements 11' and 12' respectively. This growth
`continues until the entire complementary DNA molecule has
`been constructed. Completion can be noted by identifying
`the sequence corresponding to the 8 region of template 6.
`Turning to Figure lB, a variation of this
`15 chemistry is shown in that the template 6* carries the
`reactive group X which bonds to the substrate via the A-X
`bond to form an immobilized template 5*. This is then
`hybridized with primer 3* to give the immobilized, primed
`template 9* upon which the desired adding of dNTPs takes
`20 place to add units 11 and 12 and thus identify the
`sequence and identity of units 11' and 12'. While in the
`chemistry illustrated in Figure lB reference is made to
`coupling template DNA 6* via an X group on its 3' end to
`the A group on the substrate, it will be appreciated that
`the template DNA 6* could just as well be coupled through
`its 5' end. The chemistry for such an attachment is known
`in the art.
`Referring now to Figure 2, a device 13 for
`In
`carrying ~ut the invention is shown schematically.
`this schematic representation, and the representation
`provided in Figure 2, many components such as mixers,
`valves and the like are omitted to facilitate a clear
`focus on the invention. Device 13 includes a reaction
`zone 14 which carries inside it a surface 15. A plurality
`35 of copies of a subject primed single stranded DNA are
`immobilized on this surface 15. This is the strand of DNA
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`for which the sequence is desired. The immobilized DNA is
`depicted fancifully on surface 15 as if it were present as
`a series of separately visible attached strands. As will
`be appreciated, this is not in fact the case and is only
`5 done to guide the reader as to the location of the DNA
`strands. The reaction zone 14 may be configured to permit
`direct reading of reporter signals emanating from within.
`Examples of this configuration include equipping the
`reaction zone to permit measuring fluorescence or
`luminescence through one or more transparent walls or
`detecting radionuclide decay. Reaction zone 14 is fitted
`with inlet 16 for the addition of polymerase or another
`suitable enzyme capable of moderating the templat(cid:173)
`e-directing coupling of nucleotides to one another. The
`reaction zone i~•also accessed by inlet lines, lBa-l~d for
`four differently labeled blocked dNTPs, that is
`3'blockeddA'TP, 3'blockeddC''TP, 3'blockeddG' ''TP, and
`3'blockeddT''''TP. These materials can be added in four
`separate lines, as shown, or can be premixed, if desired,
`and added via a single line. Buffer and other suitable
`reaction medium components are added via line 20.
`In practice, the polymerase and the four labeled
`dNTPs are added to the reaction zone 14 under conditions
`adequate to permit the enzyme to bring about addition of
`the one, and only the one, of the four labeled blocked
`dNTPs which is complementary to the first available
`template nucleotide following the primer. The blocking
`group present on the 3'-hydroxyl position of the added
`dNTP prevents inadvertent multiple additions. After this
`30 first addition reaction is complete, the liquid in
`reaction zone 14 is drained through line 22 either to
`waste. or if desired to storage for reuse. The reaction
`zone and the surface 15 are rinsed as appropriate to
`remove unreacted, uncoupled labeled blocked dNTPs. At
`this point the first member of the complementary chain is
`now in place associated with the subject chain attached to
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`surface 15. The identity of this first nucleotide can be
`determined by detecting and identifying the label attached
`to it.
`
`This detection and identification can be carried
`5 out in the case of a fluorescent label by irradiating the
`surface with a fluorescence-exciting beam from light
`source 24 and detecting the resulting fluorescence with
`detector 26. The detected florescence is then correlated
`to the fluorescence properties of the four different
`labels present on the four different deoxynucleotide
`triphosphates to identify exactly which one of the four
`materials was incorporated at the first position of the
`complementary chain. This identity is then noted.
`In the next step, a reaction is carried out to
`remove the blocking group and label from the 3' position
`on the first deoxynucleotide triphosphate. This reaction
`is carried out in reaction zone 14. A deblocking solution
`is added via line 28 to remove the 3' hydroxyl labeled
`blocking group. This then generates an active 3 1 hydroxyl
`20 position on the first nucleotide present in the
`complementary chain and makes it available for coupling to
`the 5' position of the second nucleotide. After
`completion of the deblocking, removal of the deblocking
`solution via line 22 and rinsing as needed, the four
`25 blocked, labeled deoxynucleotide triphosphates, buffer and
`polymerase are again added and the appropriate second
`member is then coupled into the growing complementary
`chain. Following rinsing, the second member of the chain
`can be identified based on its label.
`This process is then repeated as needed until
`the complementary chain has been completed. At the
`completion of the construction of the complementary chain,
`the seguence of incorporated deoxynucleotides is known,
`and therefore so is the sequence of the complement which
`is the subject chain.
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`It will be appreciated that this process is
`easily automated.
`It is a series of fluid additions and
`removals from a reaction zone. This can be easily
`accomplished by a series of timer-controlled valves and
`the like. This technology has been well developed in the
`area of oligonucleotide synthesizers, peptide
`synthesizers, and the like.
`In such an automated system,
`the timing can be controlled by a microprocessor or, in
`most cases, by a simple programmable timer. The rate and
`10 extent of reaction can be monitored by measurement of the
`reporter concentration at various stages.
`The labels present in the blocked dNTPs can be
`incorporated in one of several manners. For one, they can
`be incorporated directly and irremovably in the
`15 deoxynucleotide triphosphate unit itself. Thus, as the
`complementary chain grows there is a summing of signals
`and one identifies each added nucleotide by noting the
`change in signal observed after each nucleotide is added.
`Alternatively, and in many cases preferably, the
`label is incorporated within the blocking group or is
`otherwise incorporated in a way which allows it to be
`removed between each addition. This permits the detection
`to be substantially simpler in that one is noting the
`presence of one of the four reporter groups after each
`25 addition rather than a change in the sum of a group of
`reporter groups.
`In the embodiment shown in Figure 2, the
`presence of reporter signal is noted directly in the
`reaction zone 14 by the analytical system noted as source
`24 and detector 26.
`It will be appreciated, however, that
`in embodiments where the reporter group is removed during
`each cycle, it is possible to read or detect the reporter
`at a remote site after it has been carried out of the
`reaction zone 14. For example, drain line 22 could be
`35 valved to a sample collector (not shown) which would
`isolate and store the individual delabeling product
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`solutions for subsequent reading. Alternatively, if the
`nature of the label permitted, the various removed labels
`could be read as they flowed out of the reaction zone by
`equipping line 22 with an in-line measurement cell such as
`source 24' and detector 26' or the like.
`A second embodiment of this invention employs
`four separate parallel reaction zones. This method has
`the advantage of requiring only one type of labeling and
`being able to use it with all four dNTPs. Figure 3 shows
`a schematic representation of a device 30 which has the
`In this configuration
`four reaction zone configuration.
`there are four reaction zones 32a through 32d, each ·of
`In
`which resembles the reaction zone 14 in Figure 2.
`these cases each of the four reaction zones contains a
`surface 34a-d to which is immobilized numerous copies of a
`primed subject single stranded DNA. Each reaction zone is
`supplied with polymerase via lines 36a-d. Each zone is
`supplied with suitable reaction medium via lines 38a-38d.
`The four dNTPs are supplied in blocked form to each zone,
`In zone 32a one of the blocked dNTPs is labeled,
`as well.
`for example "A'"; in zone 32b a second dNTP is labeled,
`for example "C'"; in zone 32c a third dNTP is labeled, for
`example "G'"; and in 32d the fourth labeled dNTP "T'" is
`present. These labeled materials are supplied via lines
`25 40a through 40d respectively. Unlabeled blocked dNTPs are
`supplied via lines 42a-d so that each of the four reaction
`zones contains three unlabeled blocked dNTPs and one
`labeled blocked dNTP. Again, as noted with reference to
`Figure 2 ,. the various labeled and unlabeled dNTP' s can be
`30 premixed. These premixed materials can be added to the
`various reaction zones via single addition lines.
`Using the same general methodology described
`with reference to Figure 2, the single stranded DNA
`hybridized to a primer and attached to each of surfaces
`35 34a-34d is contacted with polymerase (supplied via lines
`36a-36d), buffer (supplied via lines 38a-38d) and the four
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`bases in each of the four reaction zones. The blocked
`dNTP which complements the first base on the subject chain
`couples.
`In one of the four reaction zones, this base is
`labeled. By noting in which of the four zones this label
`is incorporated into the growing chain, one can determine
`the identity of the dNTP·which is incorporated at the
`first position. This determination of the identity of the
`first unit of the chain can be carried out using signal
`sources and detectors such as 44a-44d and 46a-46d,
`respectively. Deblocking is carried out by adding
`deblocking solution to the reaction zone through lines
`48a-48d. Lines 50a-50d are drain lines for removing
`material from the reaction zones following each step.
`In this second configuration, all of the
`15 variations noted with reference to the device described in
`Figure 2 can also be used including cumulating reporter
`signals and generating reporter signals away from the
`reaction zone by removing the reporter groups as part of
`each of the sequential couplings. Clearly, this
`embodiment can be readily automated, as well.
`One obvious potential shortcoming of the present
`invention is that it employs a long sequence of serial
`reactions. Even if the efficiency and yield of each of
`these reactions are relatively high, the overall yield
`25 becomes the product of a large number of numbers, each of
`which is somewhat less than 1.00, and thus can become
`unacceptably low. For example if the yield of a given
`addition step is 98% and the deblocking is 98t- as well,
`the overall yield after 15 additions is 48i, after 30
`30 additions it is 23% and after 60 additions it is 5.3%.
`This limitation can be alleviated by
`periodically halting the DNA molecule growth and using th~
`sequence data obtained prior t0 halting the growth to
`externally recreate a portion of the molecule which can
`then be used as a primer for renewed DNA fabrication.
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`This process is illustrated in Figure 4. Figure
`4 shows a schematic of an automated sequencer 52 employing
`the present inventton. Sequencer 52 has a single reaction
`immobilized
`zone 14 combining the subject primed DNA,
`therein such as on surface 15. The four 3-blocked DNTP's,
`suitably detachably labeled, are fed to