US007057026B2
`
`(12) United States Patent
`Barnes et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7.057,026 B2
`Jun. 6, 2006
`
`(54) LABELLED NUCLEOTIDES
`
`(75) Inventors: Colin Barnes, Nr. Saffron Walden
`(GB); Shankar Balasubramanian, Nr.
`Saffron Walden (GB); Xiaohai Liu, Nr.
`Saffron Walden (GB); Harold
`Swerdlow, Nr. Saffron Walden (GB);
`John Milton, Nr. Saffron Walden (GB)
`
`(73) Assignee: Solexa Limited, Nr. Saffron Walden
`GB
`(GB)
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 336 days.
`
`(*) Notice:
`
`(21) Appl. No.: 10/227,131
`
`(22) Filed:
`
`Aug. 23, 2002
`
`(65)
`
`Prior Publication Data
`
`US 2003/O104437 A1
`
`Jun. 5, 2003
`
`(51) Int. Cl.
`(2006.01)
`C7H 2L/00
`(2006.01)
`C7H 2L/02
`(2006.01)
`C7H 2L/04
`(2006.01)
`CI2O I/68
`(52) U.S. Cl. ........................ 536/23.1; 435/6: 435/91.1;
`435/287.2:536/25.3: 536/26.6
`(58) Field of Classification Search .................... 435/6,
`435/91. 1, 287.2: 536/23.1, 25.3, 26.6
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,772,691 A
`5,302,509 A
`
`9, 1988 Herman
`4, 1994 Cheeseman
`
`7/1995 Hyman
`5,436,143 A
`5/1996 Hyman
`5,516,664 A
`8, 1996 Dower et al.
`5,547,839 A
`2, 1997 Hyman
`5,602,000 A
`6, 1998 Hiatt et al.
`5,763,594 A
`2f1999 Hiatt et al.
`5,872,244 A
`6,008.379 A 12, 1999 B
`www.
`enson et al.
`6,087,095 A
`7/2000 Rosenthal et al.
`6.214.987 B1
`4/2001 Hiatt et al.
`6.255.475 B1
`7/2001 Kwiatkowski
`6,664,079 B1* 12/2003 Ju et al. .................... 435,911
`FOREIGN PATENT DOCUMENTS
`WO 94/14972
`T 1994
`WO 96,07669
`3, 1996
`WO 99,05315
`2, 1999
`WO 99,57321
`11, 1999
`WOOOO2895
`1, 2000
`WOOOf 15844
`3, 2000
`WOOO,53812
`9, 2000
`WO O1/23610
`4/2001
`WO O1,57249
`8, 2001
`WO O2,29003
`4/2002
`
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`OTHER PUBLICATIONS
`Stragene Catalog 1988 p. 39.*
`J. Brunckova et al., Tetrahedron Letters, 1994, vol. 35, pp.
`6619-6622.
`S. Nishino et al., Heteroatom Chemistry, 1991, vol. 2, pp.
`187-196.
`
`(Continued)
`Primary Examiner Jezia Riley
`(74) Attorney, Agent, or Firm—Klauber & Jackson LLC
`
`ABSTRACT
`(57)
`Nucleosides and nucleotides are disclosed that are linked to
`detectable labels via a cleavable linker group.
`
`8 Claims, 6 Drawing Sheets
`
`Label Mcleayable linkerwin-WBas
`
`Cleavable linkers may include:
`Ss
`B
`C
`B}.
`s
`aso
`
`L
`
`n-
`O
`as
`S.
`R.
`R6
`
`where RandR, which may be the same or
`different are each selected from HOH, or
`any group which can be transformed into an
`OH, including a carbonyl
`
`RandR groups may include
`Y
`O
`O
`O.
`i-Rs
`R.
`R. Q RN R.
`R6
`R
`W.
`
`Y
`5Rs
`R. N.
`
`R
`
`2.
`2^o
`I. %
`R-S
`S-R
`3.
`K.
`s
`
`R3 represents one or more
`substituents independently
`selected from alkyl, alkoxy,
`amino or halogen
`
`Alternatively, cleavable linkers may
`be constructed from any labile
`functionality used on the 3'-block
`
`5
`
`R
`
`n
`o, one R16
`&n?
`o
`s
`ofors on-R, os-R, or
`
`o
`Y.
`Y
`so
`is
`SRs
`r
`r
`R R
`R. R.
`R. R.
`R
`RO RS R's RS-R.
`R6
`R.
`Rs
`
`where R4 is Hor alkyl, R is Hor
`alkyland R is alkyl, cycloalkyl,
`alkenyl, cycloalkenyl or benzyl
`and Xish, phosphate, diphosphate or triphosphate
`
`Columbia Ex. 2077
`Illumina, Inc. v. The Trustees
`of Columbia University in the
`City of New York
`IPR2020-00988, -01065,
`-01177, -01125, -01323
`
`

`

`US 7,057,026 B2
`US 7,057,026 B2
`Page 2
`Page 2
`
`OTHER PUBLICATIONS
`OTHER PUBLICATIONS
`M. Kreémerova, Coll. Czech. Chem. Commun., 1990 vol.
`M. Krecmerová, Coll. Czech. Chem. Commun., 1990 vol.
`55, pp. 2521-2536.
`55, pp. 2521-2536.
`P. J. L. M. Quaedflieg et al., Tetrahedron Letters, 1992 vol.
`P. J. L. M. Quaedflieg et al., Tetrahedron Letters, 1992 vol.
`33, pp. 3081-3084.
`33, pp. 3081-3084.
`
`J. I. Yamashita et al., Chem Pharm. Bull., 1987, vol. 35, pp.
`J. I. Yamashitaet al., Chem Pharm. Bull., 1987, vol. 35, pp.
`2373-2381.
`:
`.
`2373-2381.
`S.G. Zavgorodny et al., Nucleosides, Nucleotides and
`S.G. Zavgorodny et al., Nucleosides, Nucleotides and
`Nucleic Acids, 19(10-12), 1977-1991 (2000).
`Nucleic Acids,
`19(10-12),
`1977-1991 (2000).
`ucleic
`Acids,
`19
`)
`(
`)
`* cited by examiner
`* cited by examiner
`
`

`

`U.S. Patent
`U.S. Patent
`
`Jun. 6, 2006
`Jun. 6, 2006
`
`Sheet 1 of 6
`Sheet 1 of 6
`
`US 7.057,026 B2
`US 7,057,026 B2
`
`O H
`O
`H
`\-N 0
`O S-N O
`N
`N
`
`R
`Ry
`
`R2
`Re
`
`ink
`inkLin eTN abel
`Lin flabel
`
`0
`YN NH2
`
`O.
`
`LNxo ee
`
`R,
`
`Ro
`
`Linker Label
`inkLin eM abel
`
`Uridine C5-linker
`Uridine C5-linker
`
`Cytidine C5-linker
`Cytidine C5-linker
`
`linker-label
`Linker hel
`O
`NH2
`y
`QO.
`LNH2
`Nxo aN
`2 N N
`o
`N
`R R2 S
`R;
`LS
`R>
`
`\.
`N
`
`N7 Deazaadenosine C7-linker
`N7 Deazaadenosine C7-linker
`
`O
`0
`
`-N H
`=N
`H
`N
`N-Linker-Label
`NON(Mrtikertabe
`2.
`oF
`NS
`\
`S/
`~~”
`
`R2
`Ro
`
`XO
`XO
`
`R
`Ri
`
`linkernlabel
`Linker. Label
`O
`oO
`
`XO
`
`R,
`
`O
`oN eo
`N
`Re NyMy
`NH2
`NH»
`N7 Deazaguanosine C7-linker
`N? Deazaguanosine C7-linker
`
`O
`Oo
`
`N
`
`O cyMink
`O
`"-et
`xory
`-e
`inker-label
`oT a
`abel
`R2
`R
`Ro
`Ri
`
`Li
`
`Adenosine N6-linker
`Adenosine N6-linker
`
`Cytidine N4-linker
`Cytidine N4-linker
`
`where R and R2, which may be the
`-N
`out
`29
`where R, and Ry, which may be the
`xo wyNaN.
`Same or different, are each Selected
`of\9
`sameor different, are each selected
`XO
`2
`from H, OH, or any group which can be
`from H, OH, or any group which can be
`NN
`transformed into an OH. Suitable groups
`transformed into an OH. Suitable groups
`R2 Ns. H
`yy
`4
`Ro
`for R and R2 are described in Figure 3
`for R, and Rz are described in Figure 3
`HN Linker-Label
`HN
`Guanosine N2-linker
`Guanosine N2-linker
`
`R
`R;
`
`Fig. 1
`Fig. 1
`
`X = H, phosphate, diphosphate or triphosphate
`X =H, phosphate, diphosphate or triphosphate
`
`Linker—-Label
`
`

`

`U.S. Patent
`U.S. Patent
`
`Jun. 6, 2006
`Jun.6, 2006
`
`Sheet 2 of 6
`Sheet 2 of6
`
`US 7.057,026 B2
`US 7,057,026 B2
`
`Acid able Linkers
`Acid Labile Linkers
`
`O
`
`H
`
`O
`
`pyekNAN -ms
`-v-ve-C
`wn OMe
`O
`OMe
`2: Dialkoxybenzyl linker
`2: Dialkoxybenzy! linker
`y
`¥
`
`NH
`N
`NN
`base
`
`base
`
`O
`N
`C re
`NH
`N
`SS vac
`base
`
`O
`
`O
`l
`Dye NN-
`
`H
`
`N
`
`^-v-
`3: Sieber Linker
`3: Stieber Linker
`
`Disulfde L1nker
`Disulfide Linker
`
`D
`
`Dye
`eon
`0
`
`HN
`~~ S
`
`O
`S-N-K
`Ss
`NH
`s
`Šs
`base
`
`base
`base
`
`Et,
`
`2
`
`Et, 9 -- O
`IN0On
`s'
`AROON
`\
`i
`ON
`
`ye"
`
`1N
`DWe N
`Dye
`N
`H
`y
`
`N
`
`O
`
`4: Indole Linker
`4: Indole Linker
`
`NH
`NH
`
`Š
`base
`base
`
`O
`O
`-
`
`O
`oO
`
`N
`
`0
`
`ae QtyWs 5 [s HN
`Dye NY-v-ve C ro
`SX
`
`O
`
`5: "Butyl Sieber Linker
`5: ‘Butyl Sieber Linker
`
`Fig. 2
`
`

`

`U.S. Patent
`U.S. Patent
`
`Jun. 6, 2006
`Jun. 6, 2006
`
`Sheet 3 of 6
`Sheet 3 of 6
`
`US 7.057,026 B2
`US 7,057,026 B2
`
`
`Label M Cleavable linker
`Label ~~Cleavable linker
`
`t--B
`t—}—s
`Cleavable linkers may include:
`Cleavablelinkers may include:
`s s
`stgh
`B
`B
`O 2.
`e
`L BN
`L Bs N
`ia
`(s
`2
`A
`
`0L
`
`O
`0
`
`g
`B aN
`B 2.
`L3s
`|
`
`where R and R2, which may be the same or
`where R, and R>, which may be the same or
`different, are each selected from H, OH, or
`different, are each selected from H, OH,or
`any group which can be transformed into an
`any group which can be transformedinto an
`OH, including a carbonyl
`OH,including a carbonyl
`
`|
`R1 and R2 groups may include
`R, and R, groups mayinclude
`_
`-
`O
`YO
`n
`Oo
`“oO
`~
`O
`JRs
`JRs
`V
`O
`UkRs
`UKRs
`\
`R. O
`R N
`R
`O
`RF
`Ry N
`R.
`R
`R
`6
`R
`R
`6
`6
`6
`6
`6
`O
`
`O
`R5
`Rs
`
`n
`
`O
`s
`5
`
`O
`“o
`JRs
`ARs
`R. N.
`Ri N;
`
`SO R
`-k 3-2
`UX SALA
`
`~~
`-
`O
`oO
`JR5
`Rs
`S.
`R.
`S
`Ry
`Re
`R6
`
`™
`s
`
`-R
`-R
`
`a
`3
`3 WU:
`
`O
`O
`
`2
`Zz
`
`3
`3
`
`i
`“°
`o
`“Oo
`a
`L BeN
`SO
`O
`L B2.
`R BTR ORS OANRs Orso 07 R;
`R3 rcpresents one or more
`O
`O
`n
`NO
`R3 represents one or more
`~ R
`No
`“oO R
`substituents independently
`JRs R cks
`R;
`R J-
`substituents independently
`UXRs
`R KS
`UERs
`R KS
`selected from alkyl, alkoxy,
`4.
`KRs R4 OR R4
`4 JR
`selected from alkyl, alkoxy,
`4 UKRs
`O R
`O R
`4 UR
`RF
`RZ
`amino or halogen
`Ry
`NR RO a Rf
`“S—R
`amino or halogen
`R o RS RS R. S-R.
`6
`Re
`6
`6
`R6
`R6
`O
`Oo
`KRs
`UERs
`°S
`Ry
`S
`R.
`Ry
`n
`jR
`| DR;
`
`Alternatively, cleavable linkers may
`Alternatively, cleavable linkers may
`be constructed from any labile
`be constructed fromany labile
`functionality used on the 3'-block
`functionality used on the 3'-block
`
`N
`x
`O
`0
`
``O
`“oO
`KRs
`UKRs
`O
`: %
`Ry
`& R O
`Ps
`Rs
`
`~
`NS
`R
`| DR
`
`where R is H or alkyl, R5 is H or
`where Ryis H or alkyl, Rs is H or
`alkyl and R6 is alkyl, cycloalkyl,
`alkyl and Rg is alkyl, cycloalkyl,
`alkenyl, cycloalkenyl or benzyl
`alkenyl, cycloalkenyl or benzyl
`and X is H, phosphate, diphosphate or triphosphate
`and X is H, phosphate, diphosphateor triphosphate
`
`Fig. 3
`Fig. 3
`
`

`

`U.S. Patent
`
`Jun. 6, 2006
`
`Sheet 4 of 6
`
`US 7,057,026 B2
`
`‘o8I[0OVuldsreyLSdHSWUOOT“Fong-dLNPWINZ‘ZIO3Ws
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`
`
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`
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`
`

`

`U.S. Patent
`
`Jun. 6, 2006
`
`Sheet 5 of 6
`
`US 7,057,026 B2
`
`
`
`‘ostoHYuldney.LSdHSWOOT“FONG-dLNPWZ‘ZIO8Wus
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`
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`

`

`U.S. Patent
`
`Jun. 6, 2006
`
`Sheet 6 of 6
`
`US 7,057,026 B2
`
`
`
`
`
`(7)JoxUT]S[OPU]s[qeavayoployMON
`
`
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`

`

`1.
`1
`LABELLED NUCLEOTIDES
`LABELLED NUCLEOTDES
`
`RELATED APPLICATION
`RELATED APPLICATION
`
`US 7,057,026 B2
`US 7,057,026 B2
`
`2
`2
`where a series of incorporated labelled nucleotides is
`where a series of incorporated labelled nucleotides is
`required to produce a fluorescent “bar code'.
`required to produce a fluorescent “bar code”.
`
`SUMMARY OF THE INVENTION
`SUMMARY OF THE INVENTION
`
`In the present invention, a nucleoside or nucleotide mol
`In the present invention, a nucleoside or nucleotide mol-
`ecule is linked to a detectable label via a cleavable linker
`ecule is linked to a detectable label via a cleavable linker
`group attached to the base, rendering the molecule useful in
`group attachedto the base, rendering the molecule useful in
`techniques using Labelled nucleosides or nucleotides, e.g.,
`techniques using Labelled nucleosides or nucleotides, e.g.,
`sequencing reactions, polynucleotide synthesis, nucleic acid
`sequencing reactions, polynucleotide synthesis, nucleic acid
`amplification, nucleic acid hybridization assays, single
`amplification, nucleic acid hybridization assays,
`single
`nucleotide polymorphism studies, and other techniques
`nucleotide polymorphism studies, and other techniques
`using enzymes such as polymerases, reverse transcriptases,
`using enzymes such as polymerases, reverse transcriptases,
`terminal transferases, or other DNA modifying enzymes.
`terminal transferases, or other DNA modifying enzymes.
`The invention is especially useful in techniques that use
`The invention is especially useful in techniques that use
`Labelled dNTPs, such as nick translation, random primer
`Labelled dNTPs, such as nick translation, random primer
`labeling, end-labeling (e.g., with terminal deoxynucleotidyl
`labeling, end-labeling (e.g., with terminal deoxynucleotidyl-
`transferase), reverse transcription, or nucleic acid amplifi
`transferase), reverse transcription, or nucleic acid amplifi-
`cation. The molecules of the present invention are in contrast
`cation. The molecules of the present invention are in contrast
`to the prior art, where the label is attached to the ribose or
`to the prior art, where the label is attached to the ribose or
`deoxyribose sugar, or where the label is attached via a
`deoxyribose sugar, or where the label
`is attached via a
`non-cleavable linker.
`non-cleavable linker.
`According to a first aspect of the invention, a nucleotide
`According to a first aspect of the invention, a nucleotide
`or nucleoside molecule, or an analog thereof, has a base that
`or nucleoside molecule, or an analog thereof, has a base that
`is linked to a detectable label via a cleavable linker.
`is linked to a detectable label via a cleavable linker.
`The invention features a nucleotide or nucleoside mol-
`The invention features a nucleotide or nucleoside mol
`ecule, having a base that is linked to a detectable label via
`ecule, having a base that is linked to a detectable label via
`a cleavable linker. The base can be a purine, or a pyrimidine.
`a cleavable linker. The base can be a purine, or a pyrimidine.
`The base can be a deaZapurine. The molecule can have a
`The base can be a deazapurine. The molecule can have a
`ribose or deoxyribose sugar moiety. The ribose or deoxyri
`ribose or deoxyribose sugar moiety. The ribose or deoxyri-
`bose Sugar can include a protecting group attached via the 2
`bose sugar can include a protecting group attachedvia the 2'
`or 3' oxygen atom. The protecting group can be removed to
`or 3' oxygen atom. The protecting group can be removed to
`expose a 3'-OH. The molecule can be a deoxyribonucleotide
`expose a 3'-OH. The molecule can be a deoxyribonucleotide
`triphosphate. The detectable label can be a fluorophore. The
`triphosphate. The detectable label can be a fluorophore. The
`linker can be an acid labile linker, a photolabile linker, or can
`linker can be an acidlabile linker, a photolabile linker, or can
`contain a disulphide linkage.
`contain a disulphide linkage.
`The invention also features a method of labeling a nucleic
`The invention also features a methodoflabeling a nucleic
`acid molecule, where the method includes incorporating into
`acid molecule, where the method includes incorporating into
`the nucleic acid molecule a nucleotide or nucleoside mol-
`the nucleic acid molecule a nucleotide or nucleoside mol
`ecule, where the nucleotide or nucleoside molecule has a
`ecule, where the nucleotide or nucleoside molecule has a
`basethatis linked to a detectable label via a cleavable linker.
`base that is linked to a detectable label via a cleavable linker.
`The incorporating step can be accomplished via a terminal
`The incorporating step can be accomplished via a terminal
`transferase, a polymerase or a reverse transcriptase. The
`transferase, a polymerase or a reverse transcriptase. The
`base can be a purine, or a pyrimidine. The base can be a
`base can be a purine, or a pyrimidine. The base can be a
`deazapurine. The nucleotide or nucleoside molecule can
`deazapurine. The nucleotide or nucleoside molecule can
`have a ribose or deoxyribose sugar moiety. The ribose or
`have a ribose or deoxyribose sugar moiety. The ribose or
`deoxyribose Sugar can include a protecting group attached
`deoxyribose sugar can include a protecting group attached
`via the 2' or 3' oxygen atom. The protecting group can be
`via the 2' or 3' oxygen atom. The protecting group can be
`removed to expose a 3'-OH group. The molecule can be a
`removed to expose a 3'-OH group. The molecule can be a
`deoxyribonucleotide triphosphate. The detectable label can
`deoxyribonucleotide triphosphate. The detectable label can
`be a fluorophore. The linker can be an acid labile linker, a
`be a fluorophore. The linker can be an acid labile linker, a
`photolabile linker, or can contain a disulphide linkage. The
`photolabile linker, or can contain a disulphide linkage. The
`detectable label and/or the cleavable linker can be of a size
`detectable label and/or the cleavable linker can be of a size
`Sufficient to prevent the incorporation of a second nucleotide
`sufficient to prevent the incorporation of a second nucleotide
`or nucleoside into the nucleic acid molecule.
`or nucleoside into the nucleic acid molecule.
`In another aspect, the invention features a method for
`In another aspect, the invention features a method for
`determining the sequence of a target single-stranded poly
`determining the sequence of a target single-stranded poly-
`nucleotide, where the method includes monitoring the
`nucleotide, where the method includes monitoring the
`sequential incorporation of complementary nucleotides,
`sequential
`incorporation of complementary nucleotides,
`where the nucleotides each have a base that is linked to a
`where the nucleotides each have a base that is linked to a
`detectable label via a cleavable linker, and where the identity
`detectable label via a cleavable linker, and wherethe identity
`of each nucleotide incorporated is determined by detection
`of each nucleotide incorporated is determined by detection
`of the label linked to the base, and subsequent removal of the
`of the labellinkedto the base, and subsequent removalof the
`label.
`label.
`The invention also features a method for determining the
`The invention also features a method for determining the
`sequence of a target single-stranded polynucleotide, where
`sequence of a target single-stranded polynucleotide, where
`the method includes: (a) providing nucleotides, where the
`the method includes: (a) providing nucleotides, where the
`
`This application claims benefit of United Kingdom Appli
`This application claims benefit of United Kingdom Appli-
`cation No. GB0129012.1, filed Dec. 4, 2001. The entire
`cation No. GB0129012.1, filed Dec. 4, 2001. The entire
`teachings of the above application are incorporated herein
`teachings of the above application are incorporated herein
`by reference.
`by reference.
`
`FIELD OF THE INVENTION
`FIELD OF THE INVENTION
`
`This invention relates to labelled nucleotides. In particu
`This invention relates to labelled nucleotides. In particu-
`lar, this invention discloses nucleotides having a removable
`lar, this invention discloses nucleotides having a removable
`label and their use in polynucleotide sequencing methods.
`label and their use in polynucleotide sequencing methods.
`
`BACKGROUND
`BACKGROUND
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`Advances in the study of molecules have been led, in part,
`Advancesin the study of molecules have been led, in part,
`by improvement in technologies used to characterise the
`by improvement in technologies used to characterise the
`molecules or their biological reactions. In particular, the
`molecules or their biological reactions. In particular,
`the
`study of the nucleic acids DNA and RNA has benefited from
`study of the nucleic acids DNA and RNA hasbenefited from
`developing technologies used for sequence analysis and the
`developing technologies used for sequence analysis and the
`study of hybridisation events.
`study of hybridisation events.
`An example of the technologies that have improved the
`An example of the technologies that have improved the
`study of nucleic acids, is the development of fabricated
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`study of nucleic acids,
`is the development of fabricated
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`arrays of immobilised nucleic acids. These arrays consist
`arrays of immobilised nucleic acids. These arrays consist
`typically of a high-density matrix of polynucleotides immo
`typically of a high-density matrix of polynucleotides immo-
`bilised onto a solid Support material. See, e.g., Fodor et al.,
`bilised onto a solid support material. See, e.g., Fodor etal.,
`Trends Biotech. 12:19–26, 1994, which describes ways of
`Trends Biotech. 12:19-26, 1994, which describes ways of
`assembling the nucleic acids using a chemically sensitized
`assembling the nucleic acids using a chemically sensitized
`glass Surface protected by a mask, but exposed at defined
`glass surface protected by a mask, but exposed at defined
`areas to allow attachment of suitably modified nucleotide
`areas to allow attachment of suitably modified nucleotide
`phosphoramidites. Fabricated arrays can also be manufac
`phosphoramidites. Fabricated arrays can also be manufac-
`tured by the technique of “spotting known polynucleotides
`tured by the technique of “spotting” known polynucleotides
`onto a solid Support at predetermined positions (e.g., Stimp
`onto a solid support at predeterminedpositions (e.g., Stimp-
`son et al., Proc. Natl. Acad. Sci. USA 92:6379–6383, 1995).
`son et al., Proc. Natl. Acad. Sci. USA 92:6379-6383, 1995).
`A further development in array technology is the attach
`A further development in array technology is the attach-
`ment of the polynucleotides to the solid support material to
`ment of the polynucleotides to the solid support material to
`form single molecule arrayS. Arrays of this type are dis
`form single molecule arrays. Arrays of this type are dis-
`closed in International Patent App. WO 00/06770. The
`closed in International Patent App. WO 00/06770. The
`advantage of these arrays is that reactions can be monitored
`advantage of these arrays is that reactions can be monitored
`at the single molecule level and information on large num
`at the single molecule level and information on large num-
`bers of single molecules can be collated from a single
`bers of single molecules can be collated from a single
`reaction.
`reaction.
`For DNA arrays to be useful, the sequences of the
`For DNA arrays to be useful,
`the sequences of the
`molecules must be determined. U.S. Pat. No. 5,302,509
`molecules must be determined. U.S. Pat. No. 5,302,509
`discloses a method to sequence polynucleotides immobil
`discloses a method to sequence polynucleotides immobil-
`ised on a solid Support. The method relies on the incorpo
`ised on a solid support. The method relies on the incorpo-
`ration of 3'-blocked bases A, G, C and T having a different
`ration of 3'-blocked bases A, G, C and T having a different
`fluorescent label to the immobilised polynucleotide, in the
`fluorescent label to the immobilised polynucleotide, in the
`presence of DNA polymerase. The polymerase incorporates
`presence of DNA polymerase. The polymerase incorporates
`a base complementary to the target polynucleotide, but is
`a base complementary to the target polynucleotide, but is
`prevented from further addition by the 3'-blocking group.
`prevented from further addition by the 3'-blocking group.
`The label of the incorporated base can then be determined
`The label of the incorporated base can then be determined
`and the blocking group removed by chemical cleavage to
`and the blocking group removed by chemical cleavage to
`allow further polymerisation to occur.
`allow further polymerisation to occur.
`Welch et al. (Chem. Eur: J. 5(3):951-960, 1999) describes
`Welchet al. (Chem. Eur. J. 5(3):951—960, 1999) describes
`the synthesis of nucleotide triphosphates modified with a
`the synthesis of nucleotide triphosphates modified with a
`3'-O-blocking group that is photolabile and fluorescent. The
`3'-O-blocking group that is photolabile and fluorescent. The
`modified nucleotides are intended for use in DNA sequenc
`modified nucleotides are intended for use in DNA sequenc-
`ing experiments. However, these nucleotides proved to be
`ing experiments. However, these nucleotides proved to be
`difficult to incorporate onto an existing polynucleotide, due
`difficult to incorporate onto an existing polynucleotide, due
`to an inability to fit into the polymerase enzyme active site.
`to an inability to fit into the polymerase enzymeactive site.
`Zhu et al. (Cytometry 28:206-211, 1997) also discloses
`Zhu et al. (Cytometry 28:206—211, 1997) also discloses
`the use of fluorescent labels attached to a nucleotide via the
`the use of fluorescent labels attached to a nucleotide via the
`base group. The labelled nucleotides are intended for use in
`base group. The labelled nucleotides are intended for use in
`fluorescence in situ hybridisation (FISH) experiments,
`fluorescence in situ hybridisation (FISH) experiments,
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`

`

`US 7,057,026 B2
`US 7,057,026 B2
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`DECRIPTION OF THE DRAWINGS
`DECRIPTION OF THE DRAWINGS
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`The nucleotides/nucleosides are suitable for use in many
`The nucleotides/nucleosides are suitable for use in many
`different DNA-based methodologies, including DNA syn
`different DNA-based methodologies, including DNA syn-
`thesis and DNA sequencing protocols.
`thesis and DNA sequencing protocols.
`According to another aspect of the invention, a method for
`According to another aspect of the invention, a method for
`determining the sequence of a target polynucleotide com
`determining the sequence of a target polynucleotide com-
`prises monitoring the sequential incorporation of comple
`prises monitoring the sequential incorporation of comple-
`mentary nucleotides, wherein the nucleotides comprise a
`mentary nucleotides, wherein the nucleotides comprise a
`detectable label linked to the base portion of the nucleotide
`detectable label linked to the base portion of the nucleotide
`via a cleavable linker, incorporation is detected by monitor
`via a cleavable linker, incorporation is detected by monitor-
`ing the label, and the label is removed to permit further
`ing the label, and the label is removed to permit further
`nucleotide incorporation to occur.
`nucleotide incorporation to occur.
`
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`nucleotides have a basethat is linked to a detectable label via
`nucleotides have a base that is linked to a detectable label via
`a cleavable linker, and where the detectable label linked to
`a cleavable linker, and where the detectable label linked to
`each type of nucleotide can be distinguished upon detection
`each type of nucleotide can be distinguished upon detection
`from the detectable label used for other types of nucleotides:
`from the detectable label used for other types of nucleotides;
`(b) incorporating a nucleotide into the complement of the
`(b) incorporating a nucleotide into the complement of the
`target single stranded polynucleotide; (c) detecting the label
`target single stranded polynucleotide; (c) detecting the label
`of the nucleotide of (b), thereby determining the type of
`of the nucleotide of (b), thereby determining the type of
`nucleotide incorporated; (d) removing the label of the nucle
`nucleotide incorporated; (d) removingthe label of the nucle-
`otide of (b); and (e) optionally repeating steps (b)–(d) one or
`otide of (b); and (e) optionally repeating steps (b)—-(d) one or
`more times; thereby determining the sequence of a target
`more times; thereby determining the sequence of a target
`single-stranded polynucleotide.
`single-stranded polynucleotide.
`In the methods described herein, each of the nucleotides
`In the methods described herein, each of the nucleotides
`can be brought into contact with thew target sequentially,
`can be brought into contact with thew target sequentially,
`with removal of non-incorporated nucleotides prior to addi
`with removal of non-incorporated nucleotides prior to addi-
`FIG. 1 shows examplary nucleotide structures useful in
`tion of the next nucleotide, where detection and removal of
`FIG. 1 shows examplary nucleotide structures useful in
`tion of the next nucleotide, where detection and removal of
`the invention. For each structure, X can be H, phosphate,
`the label is carried out either after addition of each nucle-
`the label is carried out either after addition of each nucle
`the invention. For each structure, X can be H, phosphate,
`diphosphate or triphosphate. R and R can be the same or
`otide, or after addition of all four nucleotides.
`diphosphate or triphosphate. R, and R, can be the same or
`otide, or after addition of all four nucleotides.
`different, and can be selected from H, OH, or any group
`In the methods, all of the nucleotides can be brought into
`different, and can be selected from H, OH, or any group
`In the methods, all of the nucleotides can be brought into
`which can be transformed into an OH, including, but not
`contact with the target simultaneously, i.e., a composition
`which can be transformed into an OH, including, but not
`contact with the target simultaneously, i.e., a composition
`limited to, a carbonyl. Some Suitable functional groups for
`comprising all of the different nucleotides is brought into
`limited to, a carbonyl. Somesuitable functional groups for
`comprising all of the different nucleotides is brought into
`R and R include the structures shown in FIG. 3.
`contact with the target, and non-incorporated nucleotides are
`R, and R, include the structures shown in FIG.3.
`contact with the target, and non-incorporated nucleotides are
`FIG. 2 shows structures of linkers useful in the invention,
`FIG.2 showsstructures of linkers useful in the invention,
`removed prior to detection and subsequent to removal of the
`removedprior to detection and subsequent to removal of the
`including (1) disulfide linkers and acid labile linkers, (2)
`including (1) disulfide linkers and acid labile linkers, (2)
`label(s).
`label(s).
`dialkoxybenzyl linkers, (3) Sieber linkers, (4) indole linkers
`dialkoxybenzyl linkers, (3) Sieber linkers, (4) indole linkers
`The methods can comprise a first step and a second step,
`and (5) t-butyl Sieber linkers.
`The methods can comprisea first step and a secondstep,
`and (5) t-butyl Sieber linkers.
`where in the first step, a first composition comprising two of
`where inthefirst step, a first composition comprising two of
`FIG. 3 shows some functional molecules useful in the
`FIG. 3 shows some functional molecules useful in the
`the four nucleotides is brought into contact with the target,
`the four nucleotides is brought into contact with the target,
`invention, including some cleavable linkers and some Suit
`invention, including some cleavable linkers and somesuit-
`and non-incorporated nucleotides are removed prior to
`able hydroxyl protecting groups. In these structures, R and
`and non-incorporated nucleotides are removed prior to
`able hydroxy] protecting groups. In these structures, R, and
`detection and subsequent to removal of the label, and where
`detection and subsequent to removalof the label, and where
`R may be the same of different, and can be H, OH, or any
`R, maybe the sameof different, and can be H, OH,or any
`in the second step, a second composition comprising the two
`in the secondstep, a second composition comprising the two
`group which can be transformed into an OH group, includ
`group which can be transformed into an OH group,includ-
`nucleotides not included in the first composition is brought
`ing a carbonyl. R. represents one or more Substituents
`nucleotides not included in the first composition is brought
`ing a carbonyl. R; represents one or more substituents
`into contact with the target, and non-incorporated nucle
`independently selected from alkyl, alkoxyl, amino or halo
`into contact with the target, and non-incorporated nucle-
`independently selected from alkyl, alkoxyl, amino or halo-
`otides are removed prior to detection and Subsequent to
`gen groups. Alternatively, cleavable linkers may be con
`otides are removed prior to detection and subsequent to
`gen groups. Alternatively, cleavable linkers may be con-
`removal of the label, and where the first steps and the second
`removalofthe label, and wherethefirst steps and the second
`structed from any labile functionality used on the 3'-block.
`structed from any labile functionality used on the 3'-block.
`step can be optionally repeated one or more times.
`step can be optionally repeated one or more times.
`Ra and Rs can be H or alkyl, and R can be alkyl, cycloalkyl,
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`R, and R, can be H or alkyl, and R, can be alkyl, cycloalkyl,
`The methods described herein can also comprise a first
`alkenyl, cycloalkenyl or benzyl. X can be H. phosphate,
`The methods described herein can also compriseafirst
`alkenyl, cycloalkenyl or benzyl. X can be H, phosphate,
`step and a second step, where in the first step, a composition
`diphosphate or triphosphate.
`step and a secondstep, wherein thefirst step, a composition
`diphosphate or triphosphate.
`comprising one of the four nucleotides is brought into
`FIG. 4 shows a denaturing gel showing the incorporation
`comprising one of the four nucleotides is brought
`into
`FIG. 4 shows a denaturing gel showing the incorporation
`contact with the target, and non-incorporated nucleotides are
`of the triphosphate of Example 1 using Klenow polymerase.
`contact with the target, and non-incorporated nucleotides are
`of the triphosphate of Example 1 using Klenow polymerase.
`removed prior to detection and subsequent to removal of the
`FIG. 5 shows a denaturing gel showing the incorporation
`removedprior to detection and subsequent to removal of the
`FIG. 5 shows a denaturing gel showing the incorporation
`label, and where in the second step, a second composition
`of the triphosphate of Example 3 using Klenow polymerase.
`label, and where in the second step, a second composition
`of the triphosphate of Example 3 using Klenow polymerase.
`comprising the three nucleotides not included in the first
`FIG. 6 shows a denaturing gel showing the incorporation
`comprising the three nucleotides not included in the first
`FIG. 6 shows a denaturing gel showing the incorporation
`composition is brought into contact with the target, and
`of the triphosphate of Example 4 using Klenow polymerase.
`