United States Patent [19J
`Dower et al.
`
`[54] SEQUENCING OF SURFACE IMMOBILIZED
`POLYMERS UTILIZING
`MICROFLOURESCENCE DETECTION
`
`[75]
`
`Inventors: William J. Dower, Menlo Park;
`Stephen P. A. Fodor, Palo Alto, both
`of Calif.
`
`[73] Assignee: Affymax Technologies N.V., Curacao,
`Netherlands Antilles
`
`[21] Appl. No.: 626,730
`
`[22] Filed:
`
`Dec. 6, 1990
`
`Related U.S. Application Data
`
`[63] Continuation-in-part of Ser. No. 492,462, Mar. 7, 1990, Pat.
`No. 5,143,854, which is a continuation-in-part of Ser. No.
`362,901, Jun. 7, 1989, abandoned.
`Int. Cl.6
`[51]
`....................................................... C12Q 1/68
`[52] U.S. Cl . ........................... 435/6; 536/24.33; 536/24.3
`[58] Field of Search .................................. 435/6, 91, 810,
`435/973, 975, 91.2; 436/527, 530, 531,
`56, 94, 800, 808; 536/27, 24.33; 935/77
`
`[56]
`
`References Cited
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`4/1986 Sheldon et al .............................. 435/6
`4/1987 Mundy ........................................ 435/6
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`6/1991 Dattagupta et al. ...................... 536/27
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`6/1992 Livak et al ................................. 435/6
`9/1992 Pirrung et al ........................... 436/518
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`
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`
`I 1111111111111111 11111 lllll lllll 111111111111111 1111111111111111 Ill lllll llll
`US005547839A
`[lll Patent Number:
`[45] Date of Patent:
`
`5,547,839
`Aug. 20, 1996
`
`9013666
`5/1990 WIPO .............................. Cl2Q 1/68
`9015070 12/1990 WIPO .
`9107087
`5/1991 WIPO .
`9106678
`5/1991 WIPO .............................. C12Q 1/68
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`J. Biochem. (1989) 106:60-65.
`Ross et al., "Interstrand Crosslinks due to 4,5',8-Trimeth(cid:173)
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`201:339-351.
`Barinaga, 27 Sep. 1991, Science 253:1489.
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`Dower and Fodor, 1991, Ann. Rep. Med. Chem.
`26:271-280.
`Little, 16 Aug. 1990, Nature 346:611-612.
`Craig et al., 1990, Nucl. Acids. Res. 18:2653-2660.
`Chatterjee et al., 1990, J. Am. Chem. Soc. 112:6397-6399.
`Pfeifer et al., 10 Nov. 1989, Science 246:810-812.
`Evans et al., Jul. 1989, Proc. Natl. Acad. Sci. USA
`86:5030-4.
`Saiki and Gelfand, 1989, Amplifications 1:4-6.
`
`(List continued on next page.)
`
`Primary Examiner-W. Gary Jones
`Assistant Examiner-Scott Houtteman
`Attorney, Agent, or Finn-Townsend & Townsend & Crew
`LLP
`
`[57]
`
`ABSTRACT
`
`Means for simultaneous parallel sequence analysis of a large
`number of biological polymer macromolecules. Apparatus
`and methods may use fluorescent labels in repetitive chem(cid:173)
`istry to determine terminal monomers on solid phase immo(cid:173)
`bilized polymers. Reagents which specifically recognize
`terminal monomers are used to label polymers at defined
`positions on a solid substrate.
`
`7 Claims, 10 Drawing Sheets
`
`A
`B
`B
`A
`K)
`
`B
`
`A
`
`12
`
`14
`
`Page 1
`
`Illumina Ex. 1030
`IPR Petition - USP 10,435,742
`
`

`

`5,547,839
`Page 2
`
`OTHER PUBLICATIONS
`
`Nelson ct al., 1989, Nuc. Acids Res. 17(18):7179-7186.
`McCray ct al., 1989, Ann. Rev. Biophys. Biophys. Chem.
`18:239-270.
`Knight, 1989, Bioffech. 7:1075-1076.
`Carrano et al., 1989, Genomics 4:129-136.
`Innis ct al., Dec. 1988, Proc. Natl. Acad. Sci. USA
`85:9436-9440.
`Frank et al., Oct. 1988 Bioffech. 6:1211-1213.
`Kambara ct al., Jul. 1988, Bioffech. 6:816-821.
`Bains ct al., 1988, J. Theor. Biol. 135:303-307.
`Ye and Hong, May 1987, Scientia Sinica 30(5):503-506.
`Tabor and Richardson, Jul. 1987, Proc. Natl. Acad. Sci. USA
`84:4767-4771.
`Tabor and Richardson, 15 Nov. 1987, J. Biol. Chem.
`262:15330-15333.
`Prober ct al., 1987, Science 238:336-341.
`Michicls ct al., 1987, CABIOS 3(3):203-210.
`Coulson ct al., Oct. 1986, Proc. Natl. Acad. Sci. USA
`83:7821-5.
`Olson ct al., Oct. 1986, Proc. Natl. Acad. Sci. USA
`83:7826-7830.
`Smith et al., 12 Jun. 1986, Nature 321:674-679.
`Poustka et al., 1986, CSH Symp. Quant. Biol. 51:131-139.
`Kutateladze et al., 1986, Molekulyarnaya Biologiya
`20:267-276 (abstract only).
`Gerard et al., DNA 5(4):271-279.
`Wood et al., Mar. 1985, Proc. Natl. Acad. Sci. USA
`82:1585-1588.
`
`Smith et al., 1985, Nuc. Acids Res. 13(7):2399:2412.
`Kotewicz et al., 1985, Gene 85:249-258.
`Cimino et al., 1985, Ann. Rev. Biochem. 54:1151-1193.
`Chidgeavadze et al., Apr. 1985, FEBS Lett. 183(2):275-278.
`Chidgeavadze
`et
`al.,
`1984, Nuc. Acids Res.
`12(3): 1671-1686.
`Seed, 1982, Nuc. Acids Res. 10(5):1799-1810.
`Ruth et al., 1981, Mal. Pharm. 20:415-422.
`Maxam and Gilbert, 1980, Meth. Enz. 65:499-560.
`Parsons, 1980, Photochem, Photobiol. 32:813-821.
`Song et al., 1979, Photochem. Photobiol. 29:1177-1197.
`Houts, Feb. 1979, J. Viral. 29:517-522.
`Wiesehahn et al., Jun. 1978, Proc. Natl. Acad. Sci. USA
`75:2703-2707.
`Sanger et al., Dec. 1977, Proc. Natl. Acad. Sci. USA
`74(12):5463-5467.
`Chien et al., 1976, J. Bacterial. 127:1550-1557.
`Sanger and Coulson, 1975, J. Mo!. Biol. 94:441-448.
`Ohtsuka et al., 1974, Nuc. Acids Res. 1(10):1351-1357.
`Nossa!, 1974, J. Biol. Chem. 249(17):5668-5676.
`Jacobsen et al., 1974, Eur. J. Biochem. 45:623-627.
`Amit et al., 1974, J. Org. Chem. 39(2):192-196.
`Patchomik et al., 21 Oct. 1970, J. Am. Chem. Soc.
`92(21):6333-6335.
`Kienow and Henningsen, Jan. 1970, Proc. Natl. Acad. Sci.
`USA 65(2):168-175.
`
`Page 2
`
`

`

`U.S. Patent
`
`Aug. 20, 1996
`
`Sheet 1 of 10
`
`5,547,839
`
`A
`
`C
`
`A
`
`C
`
`A
`B
`B
`A
`K)
`
`B
`8
`A
`lm
`
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`B
`A
`A
`
`12
`
`14
`
`B *
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`A
`A
`~14
`
`l12
`
`B
`
`D
`
`FIG. L
`
`.,-16
`*
`
`A
`B
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`10
`
`12
`
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`*
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`22
`
`20
`
`8
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`A A'
`B 8'
`l,a
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`8
`8• * 26
`B
`A A'
`22
`
`20
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`A'* 24
`A
`B 8'
`18
`
`A
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`'--20 ~22
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`A A'
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`B B'
`A A'
`l22
`
`20
`
`B
`
`0
`
`FIG. 2.
`
`Page 3
`
`

`

`U.S. Patent
`
`Aug. 20, 1996
`
`Sheet 2 of 10
`
`5,547,839
`
`.
`. .
`
`0
`
`0
`
`0
`
`0
`
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`
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`
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`
`.
`(x7, Y7)
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`(x2. ,Y2)
`
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`
`.,...-C6
`
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`
`MAP
`
`_,,..-C4
`.-C3
`
`,e2
`
`.,-Cl
`
`CLUSTERS LOCALIZED AT POSITIONS
`(x1 ,YI )
`(x2 ,Y2)
`(x3,y3)
`(x4 ,y4 )
`( 151 YS)
`(xs,Ysl
`
`( X7 I Y7 )
`
`(xn,Yn>
`
`C1
`C2
`C3
`C4
`C5
`Cs
`C7
`
`en
`
`FIG. 3.
`
`Page 4
`
`

`

`U.S. Patent
`
`Aug. 20, 1996
`
`Sheet 3 of 10
`
`5,547,839
`
`A
`
`B
`
`C
`
`--
`
`-
`
`--
`
`----
`
`~
`
`o
`------
`
`---
`
`---
`
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`
`A
`
`'
`
`I OAI
`loA2
`I 0A3
`I OA4
`I OAS
`
`B
`
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`
`0 Bl
`0 82
`0~
`0 84
`0 85
`
`C
`
`gcS§
`
`0 Cl
`0 C2
`0 C3
`O C4
`0 cs
`
`FIG. 4.
`
`Page 5
`
`

`

`U.S. Patent
`
`Aug. 20, 1996
`
`Sheet 4 of 10
`
`5,547,839
`
`CLUSTER N
`
`8
`7
`6
`5
`4
`3
`2
`I
`
`2 3 4 5 6 7 8···
`
`I
`
`FIG. 5.
`
`Page 6
`
`

`

`U.S. Patent
`
`Aug. 20, 1996
`
`Sheet 5 of 10
`
`5,547,839
`
`THYMINE
`NVO~
`
`OH
`
`THYMINE
`NVO?
`
`OR
`
`- CH2 C02H
`
`yN/'-.. C02H
`0 H
`
`Page 7
`
`

`

`U.S. Patent
`
`Aug. 20, 1996
`
`Sheet 6 of 10
`
`5,547,839
`
`MULT.
`TUBE
`
`70
`
`68
`66
`
`PRE-AMP
`
`72
`
`74
`
`56
`
`COMPUTER
`
`VIDEO
`
`64
`
`LASER
`
`62
`
`58
`
`FIG. 7.
`
`Page 8
`
`

`

`U.S. Patent
`
`Aug. 20, 1996
`
`Sheet 7 of 10
`
`5,547,839
`
`SYNTHETIC SCHEME
`s·
`
`/88 _____ 86 ___ _______
`
`0 0* (I]B* []*B [TI*B
`
`8
`
`[ I J ELONGATE BY
`SINGLE NUCLEOTIDE
`
`5'
`
`NOTE:
`
`ADDS [I);
`
`!Y(W;
`5' '-90 3·
`
`[2)
`
`.
`
`S'
`
`88
`
`86
`
`3-~c60m1G1m
`
`5'
`
`.. .::---_ 8 B ~ 8
`[3J
`ETC.
`
`B
`
`ATTACH
`
`PRIMER
`
`[ll
`[3]
`
`ELONGATE BY
`SINGLE NUCLEOTIDE
`
`[2]
`
`SCAN TO DETECT
`
`[2]
`
`REMOVE BLOCKING
`& LABELING AGENT
`
`FIG. 8.
`
`RECYCLE
`
`Page 9
`
`

`

`U.S. Patent
`
`Aug. 20, 1996
`
`Sheet 8 of 10
`
`5,547,839
`
`PATHWAY TO PROTECTED NUCLEOTIDES
`
`B
`HO~
`
`OH
`
`D TMS- CL
`
`HO~-FNOC
`
`OH
`
`PREFERRED PATHWAY TO BASE PROTECTION AND FUNCTIONALIZATION
`D TBDMO-CL
`2) FMOC-CL
`
`-B-FMOC
`
`I
`
`•ro~
`
`0 Si+
`I
`
`HDAz
`
`80%
`
`B-FMOC
`HO?
`
`0 Si+
`I
`
`Na#
`
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`
`NV-0~
`
`0 Si +
`I
`
`THF
`
`BFM
`NVO~
`
`OH
`
`NHL
`
`N~
`
`o~" I
`
`FIG. 9.
`
`Page 10
`
`

`

`U.S. Patent
`
`Aug. 20, 1996
`
`Sheet 9 of 10
`
`5,547,839
`
`A
`
`B
`
`DEGRADATIVE SCHEME
`5·
`A G C
`
`POLYMER CLUSTER
`AT DISTINCT
`POSITION
`
`.--.--- *
`A G C
`
`..
`
`[I] I
`
`(2]
`
`t
`(3] !
`(4] I
`I ETC
`
`ATTACH
`I
`LABEL
`END
`I
`SCAN TO
`DISTINGUISH LABEL
`I
`REMOVE END
`
`[IJ
`[4]
`
`[2]
`
`[3]
`
`FIG.
`
`IQ
`
`RECYCLE
`
`Page 11
`
`

`

`U.S. Patent
`
`Aug. 20, 1996
`
`Sheet 10 of 10
`
`5,547,839
`
`110
`
`102
`
`DETECTOR .
`SYSTEM
`
`"-114
`
`FIG.
`
`IL
`
`Page 12
`
`

`

`5,547,839
`
`1
`SEQUENCING OF SURFACE IMMOBILIZED
`POLYMERS UTILIZING
`MICROFLOURESCENCE DETECTION
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`5
`
`This application is a continuation-in-part application of
`allowed application Ser. No. 492,462, filed Mar. 7, 1990,
`now U.S. Pat. No. 5,143,854, which is a continuation-in-part 10
`of Ser. No. 362,901, filed Jun. 7, 1989, now abandoned.
`Related applications include Ser. No. 612,671, filed Nov. 1,
`1990, now U.S. Pat. No. 5,252,743, which is a continuation(cid:173)
`in-part of Ser. No. 435,316, filed Nov. 13, 1989, now
`abandoned; Ser. No. 624,120, filed Dec. 6, 1990, now 15
`abandoned; and Ser. No. 624,114, filed Dec. 6, 1990, now
`abandoned. Each of the above is incorporated hereby refer-
`ence.
`
`2
`Ed.) Vols. 1-3, Cold Spring Harbor Press, New York, which
`is hereby incorporated herein by reference. The first method
`was developed by Maxam and Gilbert. See, e.g., Maxam and
`Gilbert (1980) "Sequencing End-Labeled DNA with Base-
`Specific Chemical Cleavages," Methods
`in Enzymol.
`65:499-560, which is hereby incorporated herein by refer(cid:173)
`ence. The polymer is chemically cleaved with a series of
`base-specific cleavage reagents thereby generating a series
`of fragments of various lengths. The various fragments, each
`resulting from a cleavage at a specific base, are run in
`parallel on a slab gel which resolves nucleic acids which
`differ in length by single nucleotides. A specific label allows
`detection of cleavages at all nucleotides relative to the
`position of the label.
`This separation requires high resolution electrophoresis or
`some other system for separating nucleic acids of very
`similar size. Thus, the target nucleic acid to be sequenced
`must usually be initially purified to near homogeneity.
`Sanger and Coulson devised two alternative methods for
`20 nucleic acid sequencing. The first method, known as the plus
`and minus method, is described in Sanger and Coulson
`(1975) J. Mot. Biol. 94:441-448, and has been replaced by
`the second method. Subsequently, Sanger and Coulson
`developed another improved sequencing method known as
`the dideoxy chain termination method. See, e.g., Sanger et
`al.
`(1977) "DNA Sequencing with Chain-Termination
`Inhibitors," Proc. NatlAcad Sci USA 74:5463-5467, which
`is hereby incorporated herein by reference This method is
`based on the inability of 2', 3' dideoxy nucleotides to be
`30 elongated by a polymerase because of the absence of a 3'
`hydroxyl group on the sugar ring, thus resulting in chain
`termination. Each of the separate chain terminating nucle(cid:173)
`otides are incorporated by a DNA polymerase, and the
`resulting terminated fragment is known to end with the
`35 corresponding dideoxy nucleotide. However, both of the
`Sanger and Coulson sequencing techniques usually require
`isolation and purification of the nucleic acid to be sequenced
`and separation of nucleic acid molecules differing in length
`by single nucleotides.
`Both the polypeptide sequencing technology and the
`oligonucleotide sequencing technologies described above
`suffer from the requirement to isolate and work with distinct
`homogeneous molecules in each determination.
`In the polypeptide technology, the terminal amino acid is
`sequentially removed and analyzed. However, the analysis is
`dependent upon only one single amino acid being removed,
`thus requiring the polypeptide to be homogeneous.
`In the case of nucleic acid sequencing, the present tech(cid:173)
`niques typically utilize very high resolution polyacrylamide
`gel electrophoresis. This high resolution separation uses
`both highly toxic acrylamide for the separation of the
`resulting molecules and usually very high voltages in run(cid:173)
`ning the electrophoresis. Both the purification and isolation
`techniques are highly tedious, time consuming and expen(cid:173)
`sive processes.
`Thus, a need exists for the capability of simultaneously
`sequencing many biological polymers without individual
`isolation and purification. Moreover, dispensing with the
`need to individually perform the high resolution separation
`of related molecules leads to greater safety, speed, and
`reliability. The present invention solves these and many
`other problems.
`
`BACKGROUND OF THE INVENTION
`
`25
`
`The present invention relates to the determination of the
`sequences of polymers immobilized to a substrate. In par(cid:173)
`ticular, one embodiment of the invention provides a method
`and apparatus for sequencing many nucleic acid sequences
`immobilized at distinct locations on a matrix surface. The
`principles and apparatus of the present invention may be
`used, for example, also in the determination of sequences of
`peptides, polypeptides, oligonucleotides, nucleic acids, oli(cid:173)
`gosaccharides, phospholipids and other biological polymers.
`It is especially useful for determining the sequences of
`nucleic acids and proteins.
`The structure and function of biological molecules are
`closely interrelated. The structure of a biological polymer,
`typically a macromolecule, is generally determined by its
`monomer sequence. For this reason, biochemists historically
`have been interested in the sequence characterization of
`biological macromolecule polymers. With the advent of
`molecular biology, the relationship between a protein
`sequence and its corresponding encoding gene sequence is
`well understood. Thus, characterization of the sequence of a 40
`nucleic acid encoding a protein has become very important.
`Partly for this reason, the development of technologies
`providing the capability for sequencing enormous amounts
`of DNA has received great interest. Technologies for this
`capability are necessary for, for example, the successful
`completion of the human genome sequencing project. Struc(cid:173)
`tural characterization of biopolymers is very important for
`further progress in many areas of molecular and cell biology.
`While sequencing of macromolecules has become 50
`extremely important, many aspects of these technologies
`have not advanced significantly over the past decade. For
`example, in the protein sequencing technologies being
`applied today the Edman degradation methods are still being
`used. See, e.g., Knight (1989) "Microsequencers for Pro- 55
`teins and Oligosaccharides," Bio/Technol. 7:1075-1076.
`Although advanced instrumentation for protein sequencing
`has been developed, see, e.g., Frank et al. (1989) "Automa(cid:173)
`tion of DNA Sequencing Reactions and Related Techniques:
`A Work Station for Micromanipulation of Liquids," Biol 60
`Technol. 6:1211-1213, this technology utilizes a homoge(cid:173)
`neous and isolated protein sample for determination of
`removed residues from that homogeneous sample.
`Likewise, in nucleic acid sequencing technology, three
`major methods for sequencing have been developed, of 65
`which two are commonly used today. See, e.g., Sambrook et
`al. (1989) Molecular Cloning: A Laboratory Manual (2d
`
`45
`
`SUMMARY OF THE INVENTION
`
`The present invention provides the means to sequence
`hundreds, thousands or even millions of biological macro-
`
`Page 13
`
`

`

`5,547,839
`
`25
`
`30
`
`3
`molecules simultaneously and without individually isolating
`each macromolecule to be sequenced. It also dispenses with
`the requirement, in the case of nucleic acids, of separating
`the products of the sequencing reactions on dangerous
`polyacrylamide gels. Adaptable to automation, the cost and 5
`effort required in sequence analysis will be dramatically
`reduced.
`This invention is most applicable, but not limited, to
`linear macromolecules. It also provides specific reagents for
`sequencing both oligonucleotides and polypeptides. It pro- 10
`vides an apparatus for automating the processes described
`herein.
`The present invention provides methods for determining
`the positions of polymers which terminate with a given
`monomer, where said polymers are attached to a surface 15
`having a plurality of positionally distinct polymers attached
`thereto, said method comprising the steps of:
`labeling a terminal monomer in a monomer type specific
`manner; and
`scanning said surface, thereby determining the positions 20
`of said label. In one embodiment, the polymers are
`polynucleotides, and usually the labeling of the termi-
`nal marker comprises incorporation of a labeled termi-
`nal monomer selected from the group of nucleotides
`consisting of adenine, cytidine, guanidine and thymi-
`dine.
`An alternative embodiment provides methods for concur(cid:173)
`rently determining which subset of a plurality of positionally
`distinct polymers attached to a solid substrate at separable
`locations terminates with a given terminal subunit, said
`method comprising the steps of:
`mixing said solid substrate with a solution comprising a
`reagent, which selectively marks positionally distinct
`polymers which terminate with said given terminal
`subunit; and
`determining with a detector which separable locations are 35
`marked, thereby determining which subset of said
`positionally distinct polymers terminated with said
`given terminal subunit. In one version, the solution
`comprises a reagent which marks the positionally dis(cid:173)
`tinct polymer with a fluorescent label moiety. In 40
`another version the terminal subunit is selected from
`the group consisting of adenosine, cytosine, guanosine,
`and thymine.
`Methods are also provided for determining which subset
`of a plurality of primer polynucleotides have a predeter- 45
`mined oligonucleotide, wherein the polynucleotides are
`complementary to distinctly positioned template strands
`which are attached to a solid substrate, said method com(cid:173)
`prising the steps of:
`selectively marking said subset of primer polynucleotides 50
`having the predetermined oligonucleotide; and
`detecting which polynucleotides are marked. In one
`embodiment, the oligonucleotide subunit is a single
`nucleotide; in another the marking comprises elongat(cid:173)
`ing said primer with a labeled nucleotide which is 55
`complementary to a template; and in a further embodi(cid:173)
`ment the marking step uses a polymerase and a blocked
`and labeled adenine.
`The invention embraces methods for concurrently obtain(cid:173)
`ing sequence information on a plurality of polynucleotides
`by use of a single label detector, said method comprising the
`steps of:
`attaching a plurality of positionally distinct polynucle(cid:173)
`otides to a solid substrate at separable locations;
`labeling said plurality of polynucleotides with a terminal
`nucleotide specific reagent, said label being detectable
`using said label detector;
`
`4
`determining whether said specific labeling reagent has
`labeled each separable location. Often, the labeling is
`performed with reagents which can distinguishably
`label alternative possible nucleotide monomers. One
`embodiment uses four replica substrates each of which
`is labeled with a specific labeling reagent for adenine,
`cytosine, guanine, or thymine. Usually, the labeling and
`determining steps are performed in succession using
`reagents specific for each of adenine, cytosine, guanine,
`and thymine monomers.
`An alternative embodiment provides methods for concur(cid:173)
`rently obtaining sequence information on a plurality of
`polynucleotides, said method comprising the steps of:
`attaching distinct polynucleotides to a plurality of distinct
`solid substrates;
`labeling said plurality of solid substrates with a terminal
`nucleotide specific labeling reagent; and
`determining whether said specific labeling reagent has
`labeled each distinct substrate. The method can be
`performed using a continuous flow of distinct solid
`substrates through a reaction solution.
`A method is provided for simultaneously sequencing a
`plurality of polymers made up of monomer units, said
`plurality of polymers attached to a substrate at definable
`positions, said method comprising the steps of:
`mixing said substrate with a reagent which specifically
`recognizes a terminal monomer, thereby providing
`identification among various terminal monomer units;
`and
`scanning said substrate to distinguish signals at definable
`positions on said substrate; and
`correlating said signals at defined positions on said sub-
`strate to provide sequential series of sequence deter(cid:173)
`minations. Often, the plurality of polymers are synthe(cid:173)
`sized by a plurality of separate cell colonies, and the
`polymers may be attached to said substrate by a car-
`bonyl linkage. In one embodiment, the polymers are
`polynucleotides, and often the substrate comprises sili(cid:173)
`con. The scanning will often identify a fluorescent
`label. In one embodiment, the reagent exhibits speci(cid:173)
`ficity of removal of terminal monomers, in another, the
`reagent exhibits specificity of labeling of terminal
`monomers.
`The invention also embraces methods for sequencing a
`plurality of distinctly positioned polynucleotides attached to
`a solid substrate comprising the steps of:
`hybridizing complementary primers to said plurality of
`polynucleotides;
`elongating a complementary primer hybridized to a poly(cid:173)
`nucleotide by adding a single nucleotide; and
`identifying which of said complementary primers have
`incorporated said nucleotide. In some versions, the
`elongating step is performed simultaneously on said
`plurality of polynucleotides linked to said substrate.
`Typically, the substrate is a two dimensional surface
`and the identifying results from a positional determi(cid:173)
`nation of the complementary primers incorporating the
`single defined nucleotide. A silicon substrate is useful
`in this method.
`Methods, are provided where the linking is by photo(cid:173)
`crosslinking polynucleotide to said complementary primer,
`where said primer is attached to said substrate. The elon(cid:173)
`gating will be often catalyzed by a DNA dependent poly-
`65 merase. In various embodiments, a nucleotide will have a
`removable blocking moiety to prevent further elongation,
`e.g., NVOC.
`
`60
`
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`5,547,839
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`6
`
`5
`A nucleotide with both a blocking moiety and labeling
`moiety will be often used.
`A further understanding of the nature and advantages of
`the invention herein may be realized by reference to the
`remaining portions of the specification and the attached 5
`drawings.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`10
`
`FIGS. lA-D illustrates a simplified and schematized
`embodiment of a degradative scheme for polymer sequenc(cid:173)
`ing.
`FIGS. 2A-D illustrates a simplified and schematized
`embodiment of a synthetic scheme for polymer sequencing.
`FIG. 3 illustrates a coordinate mapping system of a petri
`plate containing colonies. Each position of a colony can be
`assigned a distinct coordinate position.
`FIGS. 4A-C illustrates various modified embodiments of
`the substrates.
`FIGS. SA-B illustrates an idealized scanning result cor(cid:173)
`responding to a particular colony position.
`FIG. 6 illustrates particular linkers useful for attaching a
`nucleic acid to a silicon substrate. Note that thymine may be
`substituted by adenine, cytidine, guanine, or uracil.
`FIG. 7 illustrates an embodiment of the scanning system
`and reaction chamber.
`FIG. 8 illustrates the application of the synthetic scheme
`for sequencing as applied to a nucleic acid cluster localized
`to a discrete identified position. FIG. 8A illustrates sche(cid:173)
`matically, at a molecular level, the sequence of events which
`occur during a particular sequencing cycle. FIG. 8B illus(cid:173)
`trates, in a logic flow chart, how the scheme is performed.
`FIG. 9 illustrates the synthesis of a representative nucle(cid:173)
`otide analog useful in the synthetic scheme. Note that the
`FMOC may be attached to adenine, cystosine, or guanine.
`FIG. 10 illustrates the application of the degradative
`scheme for sequencing as applied to a nucleic acid cluster
`localized to a discrete identified position. FIG. lOA illus(cid:173)
`trates schematically, at a molecular level, the sequence of
`events which occur during a particular sequencing cycle.
`FIG. lOB illustrates in a logic flow chart how the scheme is
`performed.
`FIG. 11 illustrates a functionalized apparatus for perform- 45
`ing the scanning steps and sequencing reaction steps.
`
`1. Synthetic cycles
`a. synthetic scheme
`b. blocking groups
`2. Degradative cycles
`3. Conceptual principles
`E. Label
`1. Attachment
`2. Mode of detection
`F. Utility
`II. Specific Embodiments
`A. Synthetic method
`B. Chain degradation method
`III. Apparatus
`I. Sequencing Procedure for a Generic Polymer
`The present invention provides methods and apparatus for
`the preparation and use of a substrate having a plurality of
`polymers with various sequences where each small defined
`contiguous area defines a small cluster of homogeneous
`polymer sequences. The invention is described herein pri-
`20 marily with regard to the sequencing of nucleic acids but
`may be readily adapted to the sequencing of other polymers,
`typically linear biological macromolecules. Such polymers
`include, for example, both linear and cyclical polymers or
`nucleic acids, polysaccharides, phospholipids, and peptides
`25 having various different amino acids, heteropolymers in
`which the polymers are mixed, polyurethanes, polyesters,
`polycarbonates, polyureas, polyamides, polyethyleneimines,
`polyarylene sulfides, polysiloxanes, polyimides, polyac(cid:173)
`etates or mixed polymers of various sorts. In a preferred
`30 embodiment, the present invention is described in the use of
`sequencing nucleic acids.
`Various aspects of the patents and applications in the cross
`reference above are applicable to the substrates and matrix
`materials described herein, to the apparatus used for scan-
`35 ning the matrix arrays, to means for automating the scanning
`process, and to the linkage of polymers to a substrate.
`A. Overview
`The present invention is based, in part, on the ability to
`perform a step wise series of reactions which either extend
`40 or degrade a polymer by defined units.
`FIG. 1 schematizes a simplified linear two monomer
`polymer made up of A type and B type subunits. A degra(cid:173)
`dative scheme is illustrated. Panel A depicts a matrix with
`two different polymers located at positions 10 and 14, but
`with no polymer linked at position 12. A reaction is
`employed to label all of these polymers at the terminus
`opposite the attachment of the monomer. Panel B illustrates
`a label (designated by an asterisk) incorporated at position
`16 on the terminal monomers. A scan step is performed to
`locate positions 10 and 14 where polymers have been linked,
`but no polymer is located at position 12. The entire matrix
`is exposed to a reagent which is specific for removing single
`terminal A monomers, which are also labeled. The reagent is
`selected to remove only a single monomer; it will not
`remove further A monomers. Removal of the labeled A
`monomer leaves a substrate as illustrated in panel C. A scan
`step is performed and compared with the previous scan,
`indicating that the polymer located at position 12 has lost its
`label, i.e, that polymer at 12 terminated with an A monomer.
`60 The entire matrix is then exposed to a second reagent which
`is specific for removing terminal B monomers which are
`also labeled. Note that only a single B on each monomer is
`removed and that successive B monomers are not affected.
`Removal of the labeled B monomer leaves a substrate as
`illustrated in panel D. Another scan step is performed,
`indicating that the polymer located at position 14 has lost its
`label, i.e., it terminated with a B monomer. The sequence of
`
`15
`
`DETAILED DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`
`I. Sequencing Procedure for a Generic Polymer
`A. Overview
`1. Substrate and matrix
`2. Scanning system
`3. Synthetic/degradative cycles
`4. Label
`5. Utility
`B. Substrate/Matrix
`1. Non-distortable
`2. Attachment of polymer
`C. Scanning system
`1. Mapping to distinct position
`2. Detection system
`3. Digital or analog signal
`D. Synthetic or degradative cycle
`
`50
`
`55
`
`65
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`7
`treatments and scans is repeated to determine the successive
`monomers. It will be recognized that if the labeled A and B
`are distinguishable, i.e., the label on polymers at sites 10 and
`14 may be distinguished, a single removal step can be
`performed to convert the substrate as illustrated in panel B 5
`directly to that illustrated in panel D.
`An alternative embodiment employs synthetic reactions
`where a synthetic product is made at the direction of the
`attached polymer. The method is useful in the synthesis of a
`complementary nucleic acid strand by elongation of a primer
`as directed by the attached polymer.
`FIG. 2 illustrates a similar simplified polymer scheme,
`where the A and B monomer provide a complementary
`correspondence to A' and B' respectively. Thus, an A mono(cid:173)
`mer directs synthetic addition of an A' monomer and a B
`monomer directs synthetic addition of a B' monomer. Panel
`A depicts monomers attached at locations 18 and 22, but not
`at location 20. Each polymer already has one corresponding
`complementary monomer A'. The matrix, with polymers, is
`subjected to an elongation reaction which incorporates, e.g.,
`single labeled A' monomers 24 but not B' monomers, as
`depicted in panel B. The label is indicated by the asterisk.
`Note that only one A monomer is added. A scan step is
`performed to determine whether polymers located at posi(cid:173)
`tions 18 or 22 have incorporated the labeled A' monomers.
`The polymer at position 18 has, while the polymer at
`position 22 has not. Another elongation reaction which
`incorporates labeled B' monomers 26 is performed resulting
`in a matrix as depicted in panel C. Again note that only one,
`and not successive B' monomers, is added. Another scan is
`performed to determine whether a polymer located at sites
`18 or 22 has incorporated a labeled B' monomer, and the
`result indicates that the polymer located at site 22 has
`incorporated the labeled B' monomer. A next step removes
`all of the labels to provide a substrate as depicted in panel
`D. As before, if the polymer which incorporated a labeled A'
`monomer is distinguishable from a polymer which incorpo(cid:173)
`rated a labeled B' monomer, the separate elongation reac(cid:173)
`tions may be combined producing a panel C type matrix
`directly from a panel A type matrix and the scan procedure
`can distinguish which terminal monomer was incorporated.
`It will be appreciated that the process may be applied to
`more complicated polymers having more different types of
`monomers. Also, the number of scan steps can be minimized
`if the various possible labeled monomers can be differenti(cid:173)
`ated by the detector system.
`Typically, the units will be single monomers, though
`under certain circumstances the units may comprise dimers,
`trimers, or longer segments of defined length. In fact, under
`certain circumstances, the method may be operable in
`removing or adding different sized units so long as the units
`are distinguishable. However, it is very important that the
`reagents used do not remove or add successive monomers.
`This is achieved in the degradative method by use of highly
`specific reagents. In the synthetic mode, this is often
`achieved with removable blocking