I 1111111111111111 11111 111111111111111 IIIII 111111111111111 IIIIII IIII 11111111
`US007270951Bl
`
`c12) United States Patent
`Stemple et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,270,951 Bl
`Sep.18,2007
`
`(54) METHOD FOR DIRECT NUCLEIC ACID
`SEQUENCING
`
`(75)
`
`Inventors: Derek L. Stemple, Hertfordshire (GB);
`Niall A. Armes, Cambridge (GB)
`
`(73) Assignee: ASM Scientific, Inc., Cambridge, MA
`(US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by O days.
`
`(21) Appl. No.:
`
`09/936,095
`
`(22) PCT Filed:
`
`Mar. 10, 2000
`
`(86) PCT No.:
`
`PCT /GB00/00873
`
`§ 371 (c)(l),
`(2), ( 4) Date:
`
`Jun. 6, 2002
`
`(87) PCT Pub. No.: WO00/53805
`
`PCT Pub. Date: Sep. 14, 2000
`
`Related U.S. Application Data
`
`(63) Continuation-in-part of application No. 09/266,187,
`filed on Mar. 10, 1999.
`
`(51)
`
`Int. Cl.
`C12Q 1168
`(2006.01)
`C12P 19134
`(2006.01)
`C07H 21102
`(2006.01)
`C07H 21104
`(2006.01)
`(52) U.S. Cl. ........................ 435/6; 435/91.2; 536/23.1;
`536/24.3
`(58) Field of Classification Search .................... 435/6,
`435/91.2; 536/23.1, 24.3
`See application file for complete search history.
`
`(56)
`
`References Cited
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`
`WO90/13666
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`
`(Continued)
`
`Primary Examiner-Teresa E. Strzelecka
`(74) Attorney, Agent, or Firm-Mintz, Levin, Cohn, Ferris,
`Glovsky and Popeo, P.C.
`
`(57)
`
`ABSTRACT
`
`The present invention provides a novel sequencing appara(cid:173)
`tus and the methods employed to determine the nucleotide
`sequence of many single nucleic acid molecules simulta(cid:173)
`neously, in parallel. The methods and apparatus of the
`present invention offer a rapid, cost effective, high through(cid:173)
`put method by which nucleic acid molecules from any
`source can be readily sequenced without the need for prior
`amplification of the sample or prior knowledge of any
`sequence information.
`
`GB
`
`2 102 005 A
`
`1/1983
`
`13 Claims, 8 Drawing Sheets
`
`. - - - - I - -
`
`Illumina Ex. 1099
`IPR Petition - USP 10,435,742
`
`

`

`US 7,270,951 Bl
`Page 2
`
`OTHER PUBLICATIONS
`
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`a Myosin-GFP Fusion Protein Expressed In Vitro." FEBS Lett. 407:
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`3'-O-Modified-dNTP Syntheses
`by Enzymatic Mop-Up."
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`Variants by Automated DNA Sequencing with BODIPY® Dye(cid:173)
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`
`Pierce et al. "Imaging Individual Green Fluorescent Proteins."
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`(1995).
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`Enzymatic Reactions Achieved by Objective-Type Total Internal
`Reflection Fluorescence Microscopy." Biochem. Biophys. Res.
`Comm. 235: 47-53 (1997).
`Venter et al. "Shotgun Sequencing of the Human Genome." Science
`280:1540-1545 (1998).
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`RNA Polymerase." Science 282: 902-907 (1998).
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`1653-1657 (1995).
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`Polymerase Accompany Formation of a Paused Transcription Com(cid:173)
`plex"; Cell, 81:341-350 (1995).
`
`* cited by examiner
`
`Illumina Ex. 1006
`IPR Petition - USP 9,718,852
`
`

`

`Triphosphatc ~
`
`Fluorochrome
`
`~ ~ ;hotolabile linker
`Tripbosphat~ ID
`' Base
`
`'
`
`Ribose
`
`I
`
`B.
`
`11D
`
`' Base
`' Ribose
`
`Fluorochrome
`
`'
`
`A.
`
`C.
`
`FIG. 1 Example Modified Nucleotides for DNAS
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`rJJ
`('D
`
`'? ....
`
`~CIO
`N
`0
`0
`-....J
`
`('D
`
`rJJ =-('D
`.....
`....
`0 ....
`
`CIO
`
`d r.,;_
`-....l
`'N
`-....l = \0
`"'""' = "'""'
`
`UI
`
`

`

`J1!f,
`
`!Iv A-~ laser
`
`Excitation 532nm
`
`Step 1:
`Incorporation
`of nucleotide
`
`Step 2:
`Detection of label
`
`,,:!:~
`
`~
`
`Photoi~~is <360nm \>
`
`Step 3:
`Unblocking
`of nucleotide
`and removal
`of label
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`rJJ
`('D
`
`'? ....
`
`~CIO
`N
`0
`0
`-....J
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`N
`0 ....
`
`CIO
`
`FIG. 2 The DNAS Reaction Cycle
`
`d r.,;_
`-....l
`'N
`-....l = \0
`"'""' = "'""'
`
`UI
`
`

`

`oliogonuclcotide
`primer
`
`.
`
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`
`llf~ ✓'~~
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`
`rJ'1
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`
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`N
`0
`0
`--:i
`
`DNA Sample being
`
`sequenced I
`
`!,
`'
`·, ''
`' 1
`:,
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`~/ ---r.;y,_-J ¼ -;-., .~ ; ;\ ;: {?}ti;~} { \ 1)\e-if
`1JL~ \ \\1>· v \j
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`, ,-~. ·,
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`~
`i, J,\7
`\\ -~tc'i1 ...
`,~'\-
`/J'
`ilj:_
`__ JI_,,
`hexahistadine_,,,..--T. ~ Ni2+
`
`,,.-~ ~'f
`
`-
`
`DNA Polymerase
`
`Etc1'ed I derivatized spot----,.. T ~ nitrilotriacetic acid
`
`r!}!:
`
`Reaction chamber lower slide
`
`i'
`
`FIG. 3 Example of a DNAS Reaction Ce11ter
`
`('D
`('D
`
`~
`
`rJ'1 =(cid:173)
`.....
`0 ....
`
`CIO
`
`d r.,;_
`-...,l
`'N
`-...,l = \0
`""'"' = ""'"'
`
`UI
`
`

`

`Buffer inflow
`
`Upper slide
`
`Objective lens
`
`Buffer outflow
`
`Side view
`
`Reaction center array etched onto lower slide
`
`Chamber seal
`
`-:r== Buffer outflow
`
`Top vie\\'
`
`0
`
`0
`
`Buffer inflow
`
`FIG. 4 DNAS Reaction chan1ber assembly
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`rJJ
`('D
`
`'? ....
`
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`N
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`0
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`
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`('D a
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`
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`
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`
`d r.,;_
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`
`UI
`
`

`

`Microscope Field
`
`(cid:127)
`
`• (cid:127)
`•
`(cid:127) •
`(cid:127)
`• •
`•
`
`•
`
`(cid:127)
`(cid:127)
`
`(cid:127)
`
`•
`
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`
`• •
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`i•L I
`(cid:127)
`(cid:127)
`(cid:127)
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`(cid:127)
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`(cid:127)
`(cid:127)
`
`•
`
`(cid:127)
`
`(cid:127)
`
`•
`(cid:127)
`
`I
`
`I I
`lµm
`. .
`
`Size of Smgle Pixel
`
`J1<
`
`1 ~tin
`1 OX 10 pixels imaging
`one reaction center
`FIG. 5 Diagram of DNAS Reaction Center Array
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
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`('D
`
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`
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`
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`
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`.....
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`
`CIO
`
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`-....l = \0
`"'""' = "'""'
`
`UI
`
`I
`
`>I
`
`

`

`Microscope
`Objective Lens
`
`Reaction chamber slides ~ .,. .,. .,. /
`upperani;
`· · · <
`_. .,. .,.
`
`,,,,_
`
`'(.
`
`'I-
`
`¥-
`
`Non-Excited Fluorochromes
`Outside of the Wave
`
`,)
`
`~\
`
`'r-1
`
`/
`
`I
`
`........
`
`e •
`
`00
`•
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`~
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`
`~ = ~
`
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`
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`
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`
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`
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`
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`
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`
`:£. ¥-
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`...
`
`..._
`
`I
`
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`
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`
`FI uorochromes
`Excited by the Wave
`
`FIG. 6 Principle of the Evanescent Wave
`
`d r.,;_
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`-....l = \0
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`
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`
`

`

`Coo led CCD camera
`
`Microchannel Plate
`Intensifier
`
`Filter wheel
`
`Objective lens ~
`
`Reaction Chamber
`Assembly
`
`Y AG laser 532nm
`
`Ar laser 488nm line
`Y AG laser 355nm
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
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`('D
`
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`
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`0
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`
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`('D
`
`rJJ =(cid:173)
`.....
`-....J
`0 ....
`
`CIO
`
`Dichroic mirrors and shutters
`to control laser illumination
`
`FIG. 7 Setup for DNAS using TIRFM
`
`d r.,;_
`-....l
`'N
`-....l = \0
`"'""' = "'""'
`
`UI
`
`

`

`r-~
`~.
`~.
`j5}.
`:Fm :~ 1~ 1~
`2 *
`*
`*
`1Em 1[§j :~
`:§1]
`:~
`1~ :~
`3 *
`Cycle4 :~ :oo :~ 1[§j
`
`X
`
`y
`
`Z
`
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`
`Z
`
`X y z
`
`X Y Z
`
`3 *
`
`3 *
`
`3
`
`*
`
`2 *
`3
`
`FIG. 8 DNAS Data Acquisition: A 3x3 Matrix Example
`
`Cycle 1
`
`Cycle 2
`
`:
`
`Cycle 3
`
`:
`
`C,
`X --y- Z
`
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`
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`
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`
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`
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`
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`
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`
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`
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`3
`
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`
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`
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`
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`
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`
`X1
`X2
`X3
`Y1
`Y2
`Y3
`21
`22
`23
`
`e
`
`ACCT
`CCGT
`TGTC
`GACG
`GGTC
`TGCA
`AAAG
`TGAA
`GTTG
`
`e •
`
`00
`•
`~
`~
`~
`
`~ =
`
`~
`
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`'?
`....
`
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`
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`
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`0
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`
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`
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`('D
`
`=-
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`....
`
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`
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`
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`-....l
`= \0
`"'""' = "'""'
`
`UI
`
`

`

`US 7,270,951 Bl
`
`1
`METHOD FOR DIRECT NUCLEIC ACID
`SEQUENCING
`
`This application is a national stage oflnternational Appli(cid:173)
`cation Number PCT /GB00/00873 filed Mar. 10, 2000 which
`is a continuation-in-part of U.S. application Ser. No. 09/266,
`187 filed Mar. 10, 1999.
`
`FIELD OF THE INVENTION
`
`The present invention relates to methods for sequencing
`nucleic acid samples. More specifically, the present inven(cid:173)
`tion relates to methods for sequencing without the need for
`amplification; prior knowledge of some of the nucleotide
`sequence to generate the sequencing primers; and the labor(cid:173)
`intensive electrophoresis techniques.
`
`BACKGROUND OF THE INVENTION
`
`5
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`2
`674-679 (1986); Luckey et al., Nucleic Acids Res 18: 4417-
`4421(1990); Dovichi, Electrophoresis 18: 2393-2399
`(1997).
`However current DNA sequencing technologies still suf-
`fer three major limitations. First, they require a large amount
`of identical DNA molecules, which are generally obtained
`either by molecular cloning or by polymerase chain reaction
`(PCR) amplification of DNA sequences. Current methods of
`detection are insensitive and thus require a minimum critical
`10 number of labeled oligonucleotides. Also, many identical
`copies of the oligonucleotide are needed to generate a
`sequence ladder. A second limitation is that current sequenc(cid:173)
`ing techniques depend on priming from sequence-specific
`oligodeoxynucleotides that must be synthesized prior to
`15 initiating the sequencing procedure. Sanger and Coulson, J.
`Mal. Biol. 94: 441-448 (1975). The need for multiple
`identical templates necessitates the synchronous priming of
`each copy from the same predetermined site. Third, current
`sequencing techniques depend on lengthy, labor-intensive
`20 electrophoresis techniques that are 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
`experiment by the resolution obtainable on the gel.
`In an effort to dispense with the need for electrophoresis
`25 techniques, a sequencing method was developed which uses
`chain terminators that can be uncaged, or deprotected, for
`further extension. See, U.S. Pat. No. 5,302,509: Metzker et
`al. Nucleic Acids Res. 22: 4259-4267 (1994). This method
`involves repetitive cycles of base incorporation, detection of
`30 incorporation, and re-activation of the chain terminator to
`allow the next cycle of DNA synthesis. Thus, by detecting
`each added base while the DNA chain is growing, the need
`for size-fractionation is eliminated. This method is never(cid:173)
`theless still highly dependent on large amounts of nucleic
`35 acid to be sequenced and the use of known sequences for
`priming the initiation of chain or growth. Moreover, this
`technique is plagued by any inefficiencies of incorporation
`and deprotection. Because incorporation and 3'-OH regen(cid:173)
`eration are not completely efficient. a pool of initially
`40 identical extending strands can rapidly become asynchro(cid:173)
`nous and sequences carmot be resolved beyond a few limited
`initial additions.
`Thus, a need still remains in the art for a rapid, cost
`effective, high throughput method for sequencing unknown
`nucleic acid samples that eliminates the need for amplifica(cid:173)
`tion; prior knowledge of some of the nucleotide sequence to
`generate sequencing primers; and labor-intensive electro(cid:173)
`phoresis techniques.
`
`The sequencing of nucleic acid samples is an important
`analytical technique in modern molecular biology. The
`development of reliable methods for DNA sequencing has
`been crucial for understanding the function and control of
`genes and for applying many of the basic techniques of
`molecular biology. These methods have also become
`increasingly important as tools in genomic analysis and
`many non-research applications, such as genetic identifica(cid:173)
`tion, forensic analysis, genetic counseling, medical diagnos(cid:173)
`tics and many others. In these latter applications, both
`techniques providing partial sequence information, such as
`fingerprinting and sequence comparisons, and techniques
`providing full sequence determination have been employed.
`See, e.g., Gibbs et al., Proc. Natl. Acad. Sci. USA 86:
`1919-1923 (1989); Gyllensten et al., Proc. Natl. Acad. Sci.
`USA 85: 7652-7656 (1988); Carrano et al., Genonmics 4:
`129-136 (1989); Caetano-Amloles et al., Mal. Gen. Genet.
`235: 157-165 (1992); Brenner and Livak, Proc. Natl. Acad.
`Sci. USA 86: 8902-8906 (1989); Green et al., PCR Methods
`and Applications 1: 77-90 (1991); and Versalovic et al.,
`Nucleic Acid Res. 19: 6823-6831 (1991).
`Most currently available DNA sequencing methods
`require the generation of a set of DNA fragments that are
`ordered by length according to nucleotide composition. The
`generation of this set of ordered fragments occurs in one of 45
`two ways: (1) chemical degradation at specific nucleotides
`using the Maxam-Gilbert method or (2) dideoxy nucleotide
`incorporation using the Sanger method. See Maxam and
`Gilbert, Proc NatlAcad Sci USA 74: 560-564 (1977); Sanger
`et al. Proc Natl Acad Sci USA 74: 5463-5467 (1977). The 50
`type and number of required steps inherently limits both the
`number of DNA segments that can be sequenced in parallel,
`and the amount of sequence that can be determined from a
`given site. Furthermore, both methods are prone to error due
`to the anomalous migration of DNA fragments in denaturing 55
`gels. Time and space limitations inherent in these eel-based
`methods have fueled the search for alternative methods.
`In an effort to satisfy the current large-scale sequencing
`demands, improvements have been made to the Sanger
`method. For example, the use of fluorescent chain termina- 60
`tors simplifies detection of the nucleotides. The synthesis of
`longer DNA fragments and improved fragment resolution
`produces more sequence information from each experiment.
`Automated analysis of fragments in gels or capillaries has
`significantly reduced the labor involved in collecting and 65
`processing sequence information. See, e.g., Prober et al.,
`Science 238: 336-341 (1987); Smith et al., Nature 321:
`
`SUMMARY OF THE INVENTION
`
`The present invention provides rapid, cost effective, high
`throughput methods for sequencing unknown nucleic acid
`samples that eliminate the need for amplification; prior
`knowledge of some of the nucleotide sequence to generate
`sequencing primers; and labor-intensive electrophoresis
`techniques. The methods of the present invention permit
`direct nucleic acid sequencing (DNAS) of single nucleic
`acid molecules.
`According to the methods of the present invention, a
`plurality of polymerase molecules is immobilized on a solid
`support through a covalent or non-covalent interaction. A
`nucleic acid sample and oligonucleotide primers are intro(cid:173)
`duced to the reaction chamber in a buffered solution con(cid:173)
`taining all four labeled-caged nucleoside triphosphate ter(cid:173)
`minators. Template-driven elongation of a nucleic acid is
`mediated by the attached polymerases using the labeled-
`
`

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`US 7,270,951 Bl
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`5
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`4
`FIG. 2 is a schematic representation of the steps of one
`cycle of direct nucleic acid sequencing, wherein step 1
`illustrates the incorporation of a labeled-caged nucleotide,
`step 2 illustrates the detection of the label, and step 3
`illustrates the unblocking of the 3'-OH cage.
`FIG. 3 is a schematic representation of a reaction center
`depicting an immobilized polymerase and a nucleic acid
`sample being sequenced.
`FIG. 4 is a schematic representation of the reaction
`10 chamber assembly that houses the array of DNAS reaction
`centers and mediates the exchange of reagents and buffer.
`FIG. 5 is a schematic representation of a reaction center
`array. The left side panel (Microscope Field) depicts the
`view of an entire array as recorded by four successive
`15 detection events (one for each of the separate fluoro(cid:173)
`chromes ). The center panel depicts a magnified view of a
`part of the field showing the spacing of individual reaction
`centers. The far right panel depicts the camera's view of a
`single reaction center.
`FIG. 6 is a schematic representation of the principle of the
`evanescent wave.
`FIG. 7 is a schematic representation of a direct nucleic
`acid sequencing set up using total internal reflection fluo(cid:173)
`rescence microscopy.
`FIG. 8 is a schematic representation of an example of a
`data acquisition algorithm obtained from a 3x3 matrix.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`3
`caged nucleoside triphosphate terminators. Reaction centers
`are monitored by the microscope system until a majority of
`sites contain immobilized polymerase bound to a nucleic
`acid template with a single incorporated labeled-caged
`nucleotide terminator. The reaction chamber is then flushed
`with a wash buffer. Specific nucleotide incorporation is then
`determined for each active reaction center. Following detec(cid:173)
`tion, the reaction chamber is irradiated to uncage the incor(cid:173)
`porated nucleotide and flushed with wash buffer once again.
`The presence of labeled-caged nucleotides is once again
`monitored before fresh reagents are added to reinitiate
`synthesis, to verify that reaction centers are successfully
`uncaged. A persistent failure of release or incorporation,
`however, indicates failure of a reaction center. A persistent
`failure of release or incorporation consists of 2-20 cycles,
`preferably 3-10 cycles, more preferably 3-5 cycles, wherein
`the presence of a labeled-caged nucleotide is detected during
`the second detection step, indicating that the reaction center
`was not successfully uncaged. The sequencing cycle out(cid:173)
`lined above is repeated until a large proportion of reaction 20
`centers fail.
`the
`in
`The differentially-labeled nucleotides used
`sequencing methods of the present invention have a detach(cid:173)
`able labeling group and are blocked at the 3' portion with a
`detachable blocking group. In a preferred embodiment, the 25
`labeling group is directly attached to the detachable 3'
`blocking group. Uncaging of the nucleotides can be accom(cid:173)
`plished enzymatically, chemically, or preferably photolyti(cid:173)
`cally, depending on the detachable linker used to link the
`labeling group and the 3' blocking group to the nucleotide. 30
`In another preferred embodiment, the labeling group is
`attached to the base of each nucleotide with a detachable
`linker rather than to the detachable 3' blocking group. The
`labeling group and the 3' blocking group can be removed
`enzymatically, chemically, or photolytically. Alternative, the
`labeling group can be removed by a different method than
`and the 3' blocking group. For example, the labeling group
`can be removed enzymatically while the 3' blocking group
`is removed chemically, or by photochemical activation.
`Many independent reactions occur simultaneously within
`the reaction chamber, each individual reaction center gen(cid:173)
`erating a few hundred, or thousands, of base pairs. This
`apparatus has the capacity to sequence in parallel thousands
`and possibly millions of separate templates from either 45
`specified or random sequence points. The combined
`sequence from each run is on the order of several million
`base-pairs of sequence and does not require amplification,
`prior knowledge of a portion of the target sequence, or
`resolution of fragments on gels or capillaries. Simple DNA 50
`preparations from any source can be sequenced with the
`apparatus and methods of the present invention.
`
`40
`
`The present invention provides a novel sequencing appa(cid:173)
`ratus and a novel sequencing method. The method of the
`present invention, referred to herein as Direct Nucleic Acid
`Sequencing (DNAS), offers a rapid, cost effective, high
`35 throughput method by which nucleic acid molecules from
`any source can be readily sequenced without the need for
`prior amplification. DNAS can be used to determine the
`nucleotide sequence of numerous single nucleic acid mol(cid:173)
`ecules in parallel.
`
`1. DNAS Reaction Center Array
`Polymerases are attached to the solid support, spaced at
`regular intervals, in an array of reaction centers, present at
`a periodicity greater than the optical resolving power of the
`microscope system. Preferably, only one polymerase mol(cid:173)
`ecule is present in each reaction center, and each reaction
`center is located at an optically resolvable distance from the
`other reaction centers. Sequencing reactions preferably
`occur in a thin aqueous reaction chamber comprising a
`sealed cover slip and an optically transparent solid support.
`Immobilization of polymerase molecules for use in
`nucleic acid sequencing has been disclosed by Densham in
`PCT application WO 99/05315. Densham describes the
`attachment of selected amino groups within the polymerase
`55 to a dextran or N-hydroxysuccinimide ester-activated sur(cid:173)
`face. WO 99/05315; EP-A-0589867; Liifas et al., Biosens.
`Bzoelectron 10: 813-822 (1995). These techniques can be
`modified in the present invention to insure that the activated
`area is small enough so that steric hindrance will prevent the
`60 attachment of more than one polymerase at any given spot
`in the array.
`The array of reaction centers containing a single poly(cid:173)
`merase molecule is constructed using lithographic tech(cid:173)
`niques commonly used in the construction of electronic
`65 integrated circuits. This methodology has been used in the
`art to construct microscopic arrays of oilgodeoxynucleotides
`and arrays of single protein motors. See, e.g., Chee et al.,
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 (Panels A-C) is a schematic representation of
`labeled-caged terminator nucleotides for use in direct
`nucleic acid sequencing. Panel A depicts a deoxyadenosine
`triphosphate modified by attachment of a photo labile linker(cid:173)
`fluorochrome conjugate to the 3' carbon of the ribose. Panel
`B depicts an alternative configuration, wherein the fluoro(cid:173)
`chrome is attached to the base of the nucleotide by way of
`a photolabile linker. Panel C depicts the four different
`nucleotides each labeled with a fluorochrome with distinct
`spectral properties, which permits the four nucleotides to be
`distinguished during the detection phase of a direct nucleic
`acid sequencing reaction cycle.
`
`

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`15
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`25
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`5
`Science 274: 610-614 (1996); Fodor et al., Nature 364:
`555-556 (1993); Fodor et al., Science 251: 767-773 (1991);
`Gushin, et al., Anal. Biochem. 250: 203-211 (1997); Kinosita
`et al., Cell 93: 21-24 (1998); Kato-Yamada et al., J. Biol.
`Chem. 273: 19375-19377 (1998); and Yasuda et al., Cell 93:
`1117-1124 (1998). Using techniques such as photolithogra(cid:173)
`phy and/or electron beam lithography [Rai-Choudhury,
`Handbook of Microlithography, Micromachining, and
`I. Microlithography, Volume
`Microfabrication. Volume
`PM39, SPIE Press (1997); Service, Science 283: 27-28
`(1999)], the substrate is sensitized with a linking group that
`allows attachment of a single modified protein. Alterna(cid:173)
`tively, an array of sensitized sites can be generated using
`thin-film technology such as Langmuir-Blodgett. See, e.g.,
`Zasadzinski et al., Science 263: 1726-1733 (1994).
`The regular spacing of proteins is achieved by attachment
`of the protein to these sensitized sites on the substrate.
`Polymerases containing the appropriate tag are incubated
`with the sensitized substrate so that a single polymerase
`molecule attaches at each sensitized site. The attachment of 20
`the polymerase can be achieved via a covalent or non(cid:173)
`covalent interaction Examples of such linkages common in
`the art include Ni2-/hexahistidine, streptavidin/biotin, avi(cid:173)
`din/biotin, glutathione S-transferase
`(GST)/glutathione,
`monoclonal antibody/antigen, and maltose binding protein/
`maltose.
`A schematic representation of a reaction center is pre(cid:173)
`sented in FIG. 3. A DNA polymerase (e.g., from Thermus
`aquaticus) is attached to a glass microscope slide. Attach(cid:173)
`ment is mediated by a hexahistidine tag on the polymerase,
`bound by strong non-covalent interaction to a Ni2
`- atom,
`which is, in turn, held to the glass by nitrilotriacetic acid and
`a linker molecule. The nitrilotriacetic acid is covalently
`linked to the glass by a linker attached by silane chemistry.
`The silane chemistry is limited to small diameter spots
`etched at evenly spaced intervals on the glass by electron
`beam lithography or photolithography. In addition to the
`attached polymerase, the reaction center includes the tem(cid:173)
`plate DNA molecule and an oligonucleotide primer both
`bound to the polymerase. The glass slide constitutes the
`lower slide of the DNAS reaction chamber.
`Housing the array of DNAS reaction centers and medi(cid:173)
`ating the exchange of reagents and buffer is the reaction
`chamber assembly. An example of DNAS reaction chamber
`assembly is illustrated in FIG. 4. The reaction chamber is a
`sealed compartment with transparent upper and lower slides.
`The slides are held in place by a metal or plastic housing,
`which may be assembled and disassembled to allow replace(cid:173)
`ment of the slides. There are two ports that allow access to
`the chamber. One port allows the input of buffer (and
`reagents) and the other port allows buffer (and reaction
`products) to be withdrawn from the chamber. The lower
`slide carries the reaction center array. In addition, a prism is
`attached to the lower slide to direct laser light into the lower
`slide at such angle as to produce total internal reflection of
`the laser light within the lower slide. This arrangement
`allows an evanescent wave to be generated over the reaction
`center array. A high numerical aperture objective lens is used
`to focus the image of the reaction center array onto the
`digital camera system. The reaction chamber housing can be 60
`fitted with heating and cooling elements, such as a Peltier
`device, to regulate the temperature of the reactions.
`By fixing the site of nucleotide incorporation within the
`optical system, sequence information can be obtained from
`many distinct nucleic acid molecules simultaneously. A 65
`diagram of the DNAS reaction center array is given in FIG.
`5. As described above, each reaction center is attached to the
`
`US 7,270,951 Bl
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`6
`lower slide of the reaction chamber. Depicted in the left side
`panel (Microscope Field) is the view of an entire array as
`recorded by four successive detection events ( one for each
`of the separate fluorochromes ). The center panel is a mag-
`5 nified view of a part of the field showing the spacing of
`individual reaction centers. Finally, the far right panel
`depicts the camera's view of a single reaction center. Each
`reaction center is assigned 100 pixels to ensure that it is truly
`isolated. The imaging area of a single pixel relative to the 1
`10 µmxl µm area allotted to each reaction center is shown. The
`density of reaction centers is limited by the