(19)
`
`(12)
`
`TEPZZ¥Z¥46 7B_T
`EP 3 034 627 B1
`
`(11)
`
`EUROPEAN PATENT SPECIFICATION
`
`(45) Date of publication and mention
`of the grant of the patent:
`30.01.2019 Bulletin 2019/05
`
`(21) Application number: 15195765.1
`
`(22) Date of filing: 05.10.2001
`
`(51)
`
`Int Cl.:
`C12Q 1/686 (2018.01)
`C12Q 1/6874 (2018.01)
`
`C12Q 1/6869 (2018.01)
`C12Q 1/6876 (2018.01)
`
`(54) MASSIVE PARALLEL METHOD FOR DECODING DNA AND RNA
`
`MASSIVES PARALLELES VERFAHREN ZUM ENTSCHLÜSSELN VON DNA UND RNA
`
`PROCÉDÉ MASSIVEMENT PARALLÈLE POUR DÉCODER L’ADN ET L’ARN
`
`(84) Designated Contracting States:
`CH DE FR GB LI
`
`(30) Priority: 06.10.2000 US 684670
`26.06.2001 US 300894 P
`
`(43) Date of publication of application:
`22.06.2016 Bulletin 2016/25
`
`(62) Document number(s) of the earlier application(s) in
`accordance with Art. 76 EPC:
`07004522.4 / 1 790 736
`01977533.7 / 1 337 541
`
`(73) Proprietor: The Trustees of Columbia University
`in the City of
`New York
`New York, NY 10027 (US)
`
`Inventors:
`(72)
`• JU, Jingyue
`Englewood Cliffs, NJ 07632 (US)
`• LI, Zengmin
`Flushing, NY 11354 (US)
`• EDWARDS, John, Robert
`Saint Louis, MO 63108 (US)
`• ITAGAKI, Yasuhiro
`New York, NY 10027 (US)
`
`(74) Representative: Pernot, Pierre et al
`Ernest Gutmann - Yves Plasseraud
`S.A.S
`88, boulevard des Belges
`69452 Lyon Cedex 06 (FR)
`
`(56) References cited:
`WO-A1-00/06770 WO-A1-00/53805
`
`WO-A1-01/92284 WO-A1-91/06678
`US-A- 6 074 823
`
`
`• METZKER M L ET AL: "TERMINATION OF DNA
`SYNTHESIS BY NOVEL
`3’-MODIFIED-DEOXYRIBONUCLEOSIDE
`5’-TRIPHOSPHATES", NUCLEIC ACIDS
`RESEARCH, INFORMATION RETRIEVAL LTD,
`GB, vol. 22, no. 20, 1 January 1994 (1994-01-01),
`pages 4259-4267, XP001068669, ISSN: 0305-1048
`• HULTMAN T ET AL: "DIRECT SOLID PHASE
`SEQUENCING OF GENOMIC AND PLASMID DNA
`USING MAGNETIC BEADS AS SOLID SUPPORT",
`NUCLEIC ACIDS RESEARCH, INFORMATION
`RETRIEVAL LTD, GB, vol. 17, no. 13, 11 July 1989
`(1989-07-11) , pages 4937-4946, XP000371656,
`ISSN: 0305-1048
`• JU JINGYUE ET AL: "Four-color DNA sequencing
`by synthesis using cleavable fluorescent
`nucleotide reversible terminators",
`PROCEEDINGS OF THE NATIONAL ACADEMY
`OF SCIENCES, NATIONAL ACADEMY OF
`SCIENCES, US, vol. 103, no. 52, 1 January 2006
`(2006-01-01), pages 19635-19640, XP002461564,
`ISSN: 0027-8424, DOI: 10.1073/PNAS.0609513103
`• MICHAEL A. JENSEN ET AL: "DMSO and Betaine
`Greatly Improve Amplification of GC-Rich
`Constructs in De Novo Synthesis", PLOS ONE,
`vol. 5, no. 6, 11 June 2010 (2010-06-11), page
`e11024, XP055265247, DOI:
`10.1371/journal.pone.0011024
`• RAJASEKHARAN PILLAI V N: "Photoremovable
`Protecting Groups in Organic Synthesis",
`SYNTHESIS, GEORG THIEME VERLAG,
`STUTTGART, DE, vol. 1, no. 1, 1 January 1980
`(1980-01-01) , pages 1-26, XP002099233, ISSN:
`0039-7881, DOI: 10.1055/S-1980-28908
`
`Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent
`Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the
`Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been
`paid. (Art. 99(1) European Patent Convention).
`
`Printed by Jouve, 75001 PARIS (FR)
`
`(Cont. next page)
`
`EP3 034 627B1
`
`Columbia Ex. 2074
`Illumina, Inc. v. The Trustees
`of Columbia University in the
`City of New York
`IPR2020-00988, -01065,
`-01177, -01125, -01323
`
`

`

`(19)
`
`(12)
`
`TEPZZ¥Z¥46 7B_T
`EP 3 034 627 B1
`
`(11)
`
`EUROPEAN PATENT SPECIFICATION
`
`(45) Date of publication and mention
`of the grant of the patent:
`30.01.2019 Bulletin 2019/05
`
`(21) Application number: 15195765.1
`
`(22) Date of filing: 05.10.2001
`
`(51)
`
`Int Cl.:
`C12Q 1/686 (2018.01)
`C12Q 1/6874 (2018.01)
`
`C12Q 1/6869 (2018.01)
`C12Q 1/6876 (2018.01)
`
`(54) MASSIVE PARALLEL METHOD FOR DECODING DNA AND RNA
`
`MASSIVES PARALLELES VERFAHREN ZUM ENTSCHLÜSSELN VON DNA UND RNA
`
`PROCÉDÉ MASSIVEMENT PARALLÈLE POUR DÉCODER L’ADN ET L’ARN
`
`(84) Designated Contracting States:
`CH DE FR GB LI
`
`(30) Priority: 06.10.2000 US 684670
`26.06.2001 US 300894 P
`
`(43) Date of publication of application:
`22.06.2016 Bulletin 2016/25
`
`(62) Document number(s) of the earlier application(s) in
`accordance with Art. 76 EPC:
`07004522.4 / 1 790 736
`01977533.7 / 1 337 541
`
`(73) Proprietor: The Trustees of Columbia University
`in the City of
`New York
`New York, NY 10027 (US)
`
`Inventors:
`(72)
`• JU, Jingyue
`Englewood Cliffs, NJ 07632 (US)
`• LI, Zengmin
`Flushing, NY 11354 (US)
`• EDWARDS, John, Robert
`Saint Louis, MO 63108 (US)
`• ITAGAKI, Yasuhiro
`New York, NY 10027 (US)
`
`(74) Representative: Pernot, Pierre et al
`Ernest Gutmann - Yves Plasseraud
`S.A.S
`88, boulevard des Belges
`69452 Lyon Cedex 06 (FR)
`
`(56) References cited:
`WO-A1-00/06770 WO-A1-00/53805
`
`WO-A1-01/92284 WO-A1-91/06678
`US-A- 6 074 823
`
`
`• METZKER M L ET AL: "TERMINATION OF DNA
`SYNTHESIS BY NOVEL
`3’-MODIFIED-DEOXYRIBONUCLEOSIDE
`5’-TRIPHOSPHATES", NUCLEIC ACIDS
`RESEARCH, INFORMATION RETRIEVAL LTD,
`GB, vol. 22, no. 20, 1 January 1994 (1994-01-01),
`pages 4259-4267, XP001068669, ISSN: 0305-1048
`• HULTMAN T ET AL: "DIRECT SOLID PHASE
`SEQUENCING OF GENOMIC AND PLASMID DNA
`USING MAGNETIC BEADS AS SOLID SUPPORT",
`NUCLEIC ACIDS RESEARCH, INFORMATION
`RETRIEVAL LTD, GB, vol. 17, no. 13, 11 July 1989
`(1989-07-11) , pages 4937-4946, XP000371656,
`ISSN: 0305-1048
`• JU JINGYUE ET AL: "Four-color DNA sequencing
`by synthesis using cleavable fluorescent
`nucleotide reversible terminators",
`PROCEEDINGS OF THE NATIONAL ACADEMY
`OF SCIENCES, NATIONAL ACADEMY OF
`SCIENCES, US, vol. 103, no. 52, 1 January 2006
`(2006-01-01), pages 19635-19640, XP002461564,
`ISSN: 0027-8424, DOI: 10.1073/PNAS.0609513103
`• MICHAEL A. JENSEN ET AL: "DMSO and Betaine
`Greatly Improve Amplification of GC-Rich
`Constructs in De Novo Synthesis", PLOS ONE,
`vol. 5, no. 6, 11 June 2010 (2010-06-11), page
`e11024, XP055265247, DOI:
`10.1371/journal.pone.0011024
`• RAJASEKHARAN PILLAI V N: "Photoremovable
`Protecting Groups in Organic Synthesis",
`SYNTHESIS, GEORG THIEME VERLAG,
`STUTTGART, DE, vol. 1, no. 1, 1 January 1980
`(1980-01-01) , pages 1-26, XP002099233, ISSN:
`0039-7881, DOI: 10.1055/S-1980-28908
`
`Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent
`Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the
`Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been
`paid. (Art. 99(1) European Patent Convention).
`
`Printed by Jouve, 75001 PARIS (FR)
`
`(Cont. next page)
`
`EP3 034 627B1
`
`

`

`EP 3 034 627 B1
`
`• RONAGHI ET AL: "A SEQUENCING METHOD
`BASED ON REAL-TIME PYROPHOSPHATE",
`SCIENCE, AMERICAN ASSOCIATION FOR THE
`ADVANCEMENT OF SCIENCE, US, vol. 281, 17
`July 1998 (1998-07-17), pages 363-365,
`XP002135869, ISSN: 0036-8075, DOI:
`10.1126/SCIENCE.281.5375.363
`
`• DATABASE INSPEC [Online] THE INSTITUTION
`OF ELECTRICAL ENGINEERS, STEVENAGE, GB;
`24 June 1994 (1994-06-24), PELLETIER H ET AL:
`"Structures of ternary complexes of rat DNA
`polymerase [beta], a DNA template-primer, and
`ddCTP", Database accession no. 4729992 &
`SCIENCE USA, vol. 264, no. 5167, 1994, pages
`1891-1903, ISSN: 0036-8075
`
`2
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`

`EP 3 034 627 B1
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`Description
`
`Background Of The Invention
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`[0001] Throughout this application, various publications are referenced in parentheses by author and year. Full citations
`for these references may be found at the end of the specification immediately preceding the claims.
`[0002] The ability to sequence deoxyribonucleic acid (DNA) accurately and rapidly is revolutionizing biology and
`medicine. The confluence of the massive Human Genome Project is driving an exponential growth in the development
`of high throughput genetic analysis technologies. This rapid technological development involving chemistry, engineering,
`biology, and computer science makes it possible to move from studying single genes at a time to analyzing and comparing
`entire genomes.
`[0003] With the completion of the first entire human genome sequence map, many areas in the genome that are highly
`polymorphic in both exons and introns will be known. The pharmacogenomics challenge is to comprehensively identify
`the genes and functional polymorphisms associated with the variability in drug response (Roses, 2000). Resequencing
`of polymorphic areas in the genome that are linked to disease development will contribute greatly to the understanding
`of diseases, such as cancer, and therapeutic development. Thus, high-throughput accurate methods for resequencing
`the highly variable intron/exon regions of the genome are needed in order to explore the full potential of the complete
`human genome sequence map. The current state-of-the-art technology for high throughput DNA sequencing, such as
`used for the Human Genome Project (Pennisi 2000), is capillary array DNA sequencers using laser induced fluorescence
`detection (Smith et al., 1986; Ju et al. 1995, 1996; Kheterpal et al. 1996; Salas-Solano et al. 1998). Improvements in
`the polymerase that lead to uniform termination efficiency and the introduction of thermostable polymerases have also
`significantly improved the quality of sequencing data (Tabor and Richardson, 1987, 1995). Although capillary array DNA
`sequencing technology to some extent addresses the throughput and read length requirements of large scale DNA
`sequencing projects, the throughput and accuracy required for mutation studies needs to be improved for a wide variety
`of applications ranging from disease gene discovery to forensic identification. For example, electrophoresis based DNA
`sequencing methods have difficulty detecting heterozygotes unambiguously and are not 100% accurate in regions rich
`in nucleotides comprising guanine or cytosine due to compressions (Bowling et al. 1991; Yamakawa et al. 1997). In
`addition, the first few bases after the priming site are often masked by the high fluorescence signal from excess dye-
`labeled primers or dye-labeled terminators, and are therefore difficult to identify. Therefore, the requirement of electro-
`phoresis for DNA sequencing is still the bottleneck for high-throughput DNA sequencing and mutation detection projects.
`[0004] The concept of sequencing DNA by synthesis without using electrophoresis was first revealed in 1988 (Hyman,
`1988) and involves detecting the identity of each nucleotide as it is incorporated into the growing strand of DNA in a
`polymerase reaction. Such a scheme coupled with the chip format and laser-induced fluorescent detection has the
`potential to markedly increase the throughput of DNA sequencing projects. Consequently, several groups have inves-
`tigated such a system with an aim to construct an ultra high-throughput DNA sequencing procedure (Cheeseman 1994,
`Metzker et al. 1994). Thus far, no complete success of using such a system to unambiguously sequence DNA has been
`reported. The pyrosequencing approach that employs four natural nucleotides (comprising a base of adenine (A), cytosine
`(C), guanine (G), or thymine (T)) and several other enzymes for sequencing DNA by synthesis is now widely used for
`mutation detection (Ronaghi 1998). In this approach, the detection is based on the pyrophosphate (PPi) released during
`the DNA polymerase reaction, the quantitative conversion of pyrophosphate to adenosine triphosphate (ATP) by sulfu-
`rylase, and the subsequent production of visible light by firefly luciferase. This procedure can only sequence up to 30
`base pairs (bps) of nucleotide sequences, and each of the 4 nucleotides needs to be added separately and detected
`separately. Long stretches of the same bases cannot be identified unambiguously with the pyrosequencing method.
`[0005] More recent work in the literature exploring DNA sequencing by a synthesis method is mostly focused on
`designing and synthesizing a photocleavable chemical moiety that is linked to a fluorescent dye to cap the 3’-OH group
`of deoxynucleoside triphosphates (dNTPs) (Welch et al. 1999). Limited success for the incorporation of the 3’-modified
`nucleotide by DNA polymerase is reported. The reason is that the 3’-position on the deoxyribose is very close to the
`amino acid residues in the active site of the polymerase, and the polymerase is therefore sensitive to modification in
`this area of the deoxyribose ring. On the other hand, it is known that modified DNA polymerases (Thermo Sequenase
`and Taq FS polymerase) are able to recognize nucleotides with extensive modifications with bulky groups such as energy
`transfer dyes at the 5-position of the pyrimidines (T and C) and at the 7-position of purines (G and A) (Rosenblum et al.
`1997, Zhu et al. 1994). The ternary complexes of rat DNA polymerase, a DNA template-primer, and dideoxycytidine
`triphosphate (ddCTP) have been determined (Pelletier et al. 1994) which supports this fact. As shown in Figure 1, the
`3-D structure indicates that the surrounding area of the 3’-position of the deoxyribose ring in ddCTP is very crowded,
`while there is ample space for modification on the 5-position the cytidine base.
`[0006] The approach disclosed in the present application is to make nucleotide analogues by linking a unique label
`such as a fluorescent dye or a mass tag through a cleavable linker to the nucleotide base or an analogue of the nucleotide
`base, such as to the 5-position of the pyrimidines (T and C) and to the 7-position of the purines (G and A), to use a small
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`EP 3 034 627 B1
`
`cleavable chemical moiety to cap the 3’-OH group of the deoxyribose to make it nonreactive, and to incorporate the
`nucleotide analogues into the growing DNA strand as terminators. Detection of the unique label will yield the sequence
`identity of the nucleotide. Upon removing the label and the 3’-OH capping group, the polymerase reaction will proceed
`to incorporate the next nucleotide analogue and detect the next base.
`[0007]
`It is also desirable to use a photocleavable group to cap the 3’-OH group. However, a photocleavable group is
`generally bulky and thus the DNA polymerase will have difficulty to incorporate the nucleotide analogues containing a
`photocleavable moiety capping the 3’-OH group. If small chemical moieties that can be easily cleaved chemically with
`high yield can be used to cap the 3’-OH group, such nucleotide analogues should also be recognized as substrates for
`DNA polymerase. It has been reported that 3’-O-methoxy-deoxynucleotides are good substrates for several polymerases
`(Axelrod et al. 1978). 3’-O-allyl-dATP was also shown to be incorporated by Ventr(exo-) DNA polymerase in the growing
`strand of DNA (Metzker et al. 1994). However, the procedure to chemically cleave the methoxy group is stringent and
`requires anhydrous conditions. Thus, it is not practical to use a methoxy group to cap the 3’-OH group for sequencing
`DNA by synthesis. An ester group was also explored to cap the 3’-OH group of the nucleotide, but it was shown to be
`cleaved by the nucleophiles in the active site in DNA polymerase (Canard et al. 1995). Chemical groups with electrophiles
`such as ketone groups are not suitable for protecting the 3’-OH of the nucleotide in enzymatic reactions due to the
`existence of strong nucleophiles in the polymerase. It is known that MOM (-CH2OCH3) and allyl (-CH2CH=CH2) groups
`can be used to cap an -OH group, and can be cleaved chemically with high yield (Ireland et al. 1986; Kamal et al. 1999) .
`The approach disclosed in the present application is to incorporate nucleotide analogues, which are labeled with cleav-
`able, unique labels such as fluorescent dyes or mass tags and where the 3’-OH is capped with a cleavable chemical
`moiety such as either a MOM group (-CH2OCH3) or an allyl group (-CH2CH=CH2), into the growing strand DNA as
`terminators. The optimized nucleotide set (3’-RO-A-LABEL1, 3’-RO-C-LABEL2, 3’-RO-G-LABEL3, 3’-RO-T-LABEL4, where R de-
`notes the chemical group used to cap the 3’-OH) can then be used for DNA sequencing by the synthesis approach.
`[0008] There are many advantages of using mass spectrometry (MS) to detect small and stable molecules. For example,
`the mass resolution can be as good as one dalton. Thus, compared to gel electrophoresis sequencing systems and the
`laser induced fluorescence detection approach which have overlapping fluorescence emission spectra, leading to het-
`erozygote detection difficulty, the MS approach disclosed in this application produces very high resolution of sequencing
`data by detecting the cleaved small mass tags instead of the long DNA fragment. This method also produces extremely
`fast separation in the time scale of microseconds. The high resolution allows accurate digital mutation and heterozygote
`detection. Another advantage of sequencing with mass spectrometry by detecting the small mass tags is that the com-
`pressions associated with gel based systems are completely eliminated.
`[0009]
`In order to maintain a continuous hybridized primer extension product with the template DNA, a primer that
`contains a stable loop to form an entity capable of self-priming in a polymerase reaction can be ligated to the 3’ end of
`each single stranded DNA template that is immobilized on a solid surface such as a chip. This approach will solve the
`problem of washing off the growing extension products in each cycle.
`[0010] Saxon and Bertozzi (2000) developed an elegant and highly specific coupling chemistry linking a specific group
`that contains a phosphine moiety to an azido group on the surface of a biological cell. In the present application, this
`coupling chemistry is adopted to create a solid surface which is coated with a covalently linked phosphine moiety, and
`to generate polymerase chain reaction (PCR) products that contain an azido group at the 5’ end for specific coupling of
`the DNA template with the solid surface. One example of a solid surface is glass channels which have an inner wall with
`an uneven or porous surface to increase the surface area. Another example is a chip.
`[0011] The present specification discloses a novel and advantageous system for DNA sequencing by the synthesis
`approach which employs a stable DNA template, which is able to self prime for the polymerase reaction, covalently
`linked to a solid surface such as a chip, and 4 unique nucleotides analogues (3’-RO-A-LABEL1, 3’-RO-C-LABEL2, 3’-RO-G-
`LABEL3, 3’-RO-T-LABEL4). The success of this novel system will allow the development of an ultra high-throughput and
`high fidelity DNA sequencing system for polymorphism, pharmacogenetics applications and for whole genome sequenc-
`ing. This fast and accurate DNA resequencing system is needed in such fields as detection of single nucleotide poly-
`morphisms (SNPs) (Chee et al. 1996), serial analysis of gene expression (SAGE) (Velculescu et al. 1995), identification
`in forensics, and genetic disease association studies.
`[0012] Stemple Derek Lyle; Armes Niall Antony (WO 00/53805 A1) discloses a sequencing apparatus and methods
`employed to determine the nucleotide sequence of many single nucleic acid molecules simultaneously. Stemple Derek
`Lyle; Armes Niall Antony does not disclose the oligonucleotides or methods of sequencing deoxyribonucleic acids of
`the current invention, which comprise nucleotide analogues having a label attached through a cleavable linker to the
`base and a small chemically cleavable chemical moiety capping the 3’-OH group.
`[0013] Balasubramanian Shankar; Klenerman David discloses a device comprising an array of molecules immobilized
`on a solid surface is disclosed, wherein the array has a surface density which allows each molecule to be individually
`resolved, e.g. by optical microscopy. Balasubramanian Shankar; Klenerman David does not disclose the oligonucleotides
`or methods of sequencing deoxyribonucleic acids of the current invention, which comprise nucleotide analogues having
`a label attached through a cleavable linker to the base and a small chemically cleavable chemical moiety capping the
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`EP 3 034 627 B1
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`3’-OH group.
`
`Summary Of The Invention
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`[0014] This invention is directed to a method for simultaneously sequencing a plurality of different deoxyribonucleic
`acids, wherein the plurality of different deoxyribonucleic acids is covalently immobilized on a solid surface, and wherein
`a sequencing method by synthesis comprising a plurality of cycles, each cycle having a plurality of steps, is simultaneously
`applied to each of said covalently immobilized different deoxyribonucleic acids, said sequencing method involving the
`detection of the identity of a plurality of nucleotide analogues incorporated into a plurality of growing strands of DNA
`hybridized to deoxyribonucleic acids, said method comprising:
`
`(a) providing to the plurality of different deoxyribonucleic acids more than one nucleotide analogue, selected from
`the group consisting of aA, aC, aG, aT, and aU, wherein each nucleotide analogue is labeled with a unique label
`attached through a cleavable linker to the base and contains a small chemically cleavable chemical moiety capping
`the 3’-OH group, wherein said small chemically cleavable chemical moiety is removable by chemical means, under
`conditions such that a plurality of growing strands are extended by incorporation of one nucleotide analogue per
`strand so as to create a plurality of extended growing strands of DNA using a DNA polymerase reaction, said
`incorporated analogues serving as terminators of the polymerase reaction;
`
`(b) detecting said unique label of said incorporated nucleotide analogues, so as to thereby identify 10,000 or more
`of the nucleotide analogues as having been incorporated into the plurality of growing strands;
`
`(c) removing the label and removing by chemical means the small chemically cleavable chemical moiety of said
`incorporated nucleotide analogues capping the 3’-OH group; and
`
`(d) repeating the cycle of steps (a) through (c);
`
`wherein the plurality of different deoxyribonucleic acids is covalently immobilized in a plurality of spots on a solid surface,
`wherein each spot comprises a plurality of the same deoxyribonucleic acid, and wherein the unique labels are dyes
`having a unique fluorescence emission, and the unique fluorescence emission from a specific dye on the dye-labeled
`nucleotide analogues on each spot of the solid surface will reveal the identity of the incorporated nucleotide; and
`wherein the small chemically cleavable chemical moiety capping the 3’-OH group:
`
`(i) is a -CH2OCH3 group or a -CH2CH=CH2 group, or is as small as a -CH2CH=CH2 group or a -CH2OCH3 group,
`(ii) does not contain a ketone group,
`(iii) when bound to the 3’-oxygen, does not form a methoxy group or an ester group, and
`(iv) forms a 3’-OH group on the deoxyribose upon cleavage of the small chemically cleavable chemical moiety
`capping the 3’-OH group; and
`
`wherein at least one of said incorporated nucleotide analogues is a 7-deaza adenine nucleotide analogue or 7-deaza
`guanine nucleotide analogue and said unique label is attached through a cleavable linker to a 7-position of deaza-
`adenine or deaza-guanine.
`[0015] This invention is also directed to a plurality of different deoxyribonucleic acids covalently immobilized on a solid
`support, said plurality of different deoxyribonucleic acids comprising incorporated nucleotide analogues, wherein each
`nucleotide analogue is labeled with a unique label attached through a cleavable linker to the base and contains a small
`chemical moiety capping its 3’-OH group, wherein said small chemically cleavable chemical moiety is removable by
`chemical means, and wherein deoxyribonucleic acids having the same sequence are immobilized at a spot and greater
`than 10,000 spots are present on the solid support;
`wherein the unique labels are dyes having a unique fluorescence emission, and the unique fluorescence emission from
`a specific dye on the dye-labeled nucleotide analogues on each spot of the solid surface will reveal the identity of the
`incorporated nucleotide; and
`wherein the small chemically cleavable chemical moiety capping the 3’-OH group:
`
`(i) is a -CH2OCH3 group or a -CH2CH=CH2 group, or is as small as a -CH2CH=CH2 group or a -CH2OCH3 group,
`
`(ii) does not contain a ketone group,
`
`(iii) when bound to the 3’-oxygen, does not form a methoxy group or an ester group, and
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`EP 3 034 627 B1
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`(iv) forms a 3’-OH group on the deoxyribose upon cleavage of the small chemically cleavable chemical moiety
`capping the 3’-OH group; and
`
`wherein at least one of said incorporated nucleotide analogues is a 7-deaza adenine nucleotide analogue or 7-deaza
`guanine nucleotide analogue and said unique label is attached through a cleavable linker to a 7-position of deaza-
`adenine or deaza-guanine.
`[0016] This invention is also directed to a method for sequencing a plurality of different deoxyribonucleic acids by
`synthesis involving the detection of the identity of nucleotide analogues incorporated into a plurality of different growing
`strands of DNA hybridized to deoxyribonucleic acids, said method comprising:
`
`(a) incorporating one nucleotide analogue into each growing strand of the plurality of different growing strands of
`DNA using a DNA polymerase reaction, wherein each nucleotide analogue is a terminator in the DNA polymerase
`reaction and wherein each nucleotide analogue comprises a unique label attached through a cleavable linker to the
`base and a small chemically cleavable chemical moiety capping the 3’-OH group that is removable by chemical
`means; and
`
`(b) detecting said label of each said incorporated nucleotide analogue, so as to thereby identify 10,000 or more of
`the incorporated nucleotide analogues, wherein said method is applied simultaneously to different deoxyribonucleic
`acids covalently immobilized on a solid surface;
`
`wherein the plurality of different deoxyribonucleic acids is covalently immobilized in a plurality of spots on a solid surface,
`wherein each spot comprises a plurality of the same deoxyribonucleic acid, and wherein the unique labels are dyes
`having a unique fluorescence emission, and the unique fluorescence emission from a specific dye on the dye-labeled
`nucleotide analogues on each spot of the solid surface will reveal the identity of the incorporated nucleotide; and
`wherein the small chemically cleavable chemical moiety capping the 3’-OH group:
`
`(i) is a -CH2OCH3 group or a -CH2CH=CH2 group, or is as small as a -CH2CH=CH2 group or a-CH2OCH3 group,
`(ii) does not contain a ketone group,
`(iii) when bound to the 3’-oxygen, does not form a methoxy group or an ester group, and
`(iv) forms a 3’-OH group on the deoxyribose upon cleavage of the small chemically cleavable chemical moiety
`capping the 3’-OH group; and
`
`wherein at least one of said incorporated nucleotide analogues is a 7-deaza adenine nucleotide analogue or 7-deaza
`guanine nucleotide analogue and said unique label is attached through a cleavable linker to a 7-position of deaza-
`adenine or deaza-guanine.
`[0017] This specification discloses a method for sequencing a nucleic acid by detecting the identity of a nucleotide
`analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction, which
`comprises the following steps:
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`(i) attaching a 5’ end of the nucleic acid to a solid surface;
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`(ii) attaching a primer to the nucleic acid attached to the solid surface;
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`(iii) adding a polymerase and one or more different nucleotide analogues to the nucleic acid to thereby incorporate
`a nucleotide analogue into the growing strand of DNA, wherein the incorporated nucleotide analogue terminates
`the polymerase reaction and wherein each different nucleotide analogue comprises (a) a base selected from the
`group consisting of adenine, guanine, cytosine, thymine, and uracil, and their analogues; (b) a unique label attached
`through a cleavable linker to the base or to an analogue of the base; (c) a deoxyribose; and (d) a cleavable chemical
`group to cap an -OH group at a 3’-position of the deoxyribose;
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`(iv) washing the solid surface to remove unincorporated nucleotide analogues;
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`(v) detecting the unique label attached to the nucleotide analogue that has been incorporated into the growing strand
`of DNA, so as to thereby identify the incorporated nucleotide analogue;
`
`(vi) adding one or more chemical compounds to permanently cap any unreacted -OH group on the primer attached
`to the nucleic acid or on a primer extension strand formed by adding one or more nucleotides or nucleotide analogues
`to the primer;
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`EP 3 034 627 B1
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`(vii) cleaving the cleavable linker between the nucleotide analogue that was incorporated into the growing strand of
`DNA and the unique label;
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`(viii) cleaving the cleavable chemical group capping the -OH group at the 3’-position of the deoxyribose to uncap
`the -OH group, and washing the solid surface to remove cleaved compounds; and
`
`(ix) repeating steps (iii) through (viii) so as to detect the identity of a newly incorporated nucleotide analogue into
`the growing strand of DNA;
`
`wherein if the unique label is a dye, the order of steps (v) through (vii) is: (v), (vi), and (vii); and
`wherein if the unique label is a mass tag, the order of steps (v) through (vii) is: (vi), (vii), and (v).
`[0018] The specification provides a method of attaching a nucleic acid to a solid surface which comprises:
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`(i) coating the solid surface with a phosphine moiety,
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`(ii) attaching an azido group to a 5’ end of the nucleic acid, and
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`(iii) immobilizing the 5’ end of the nucleic acid to the solid surface through interaction between the phosphine moiety
`on the solid surface and the azido group on the 5’ end of the nucleic acid.
`
`[0019] The specification provides a nucleotide analogue which comprises:
`
`(a) a base selected from the group consisting of adenine or an analogue of adenine, cytosine or an analogue of
`cytosine, guanine or an analogue of guanine, thymine or an analogue of thymine, and uracil or an analogue of uracil;
`
`(b) a unique label attached through a cleavable linker to the base or to an analogue of the base;
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`(c) a deoxyribose; and
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`(d) a cleavable chemical group to cap an -OH group at a 3’-position of the deoxyribose.
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`[0020] The specification provides a parallel mass spectrometry system, which comprises a plurality of atmospheric
`pressure chemical ionization mass spectrometers for parallel analysis of a plurality of samples comprising mass tags.
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`Brief Description Of The Figures
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`[0021]
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`Figure 1: The 3D structure of the ternary complexes of rat DNA polymerase, a DNA template-primer, and dideox-
`ycytidine triphosphate (ddCTP). The left side of the illustration shows the mechanism for the addition of ddCTP and
`the right side of the illustration shows the active site of the polymerase. Note that the 3’ position of the dideoxyribose
`ring is very crowded, while ample space is available at the 5 position of the cytidine base.
`
`Figure 2A-2B: Scheme of sequencing by the synthesis approach. A: Example where the unique labels are dyes
`and the solid surface is a chip. B: Example where the unique labels are mass tags and the solid surface is channels
`etched into a glass chip. A, C, G, T; nucleotide triphosphates comprising bases adenine, cytosine, guanine, and
`thymine; d, deoxy; dd, dideoxy; R, cleavable chemical group used to cap the -OH group; Y, cleavable linker.
`
`Figure 3: The synthetic scheme for the immobilization of an azido (N3) labeled DNA fragment to a solid surface
`coated with a triarylphosphine moiety. Me, methyl group; P, phosphorus; Ph, phenyl.
`
`Figure 4: The synthesis of triarylphosphine N-hydroxysuccinimide (NHS) ester.
`
`Figure 5: The synthetic scheme for attaching an azido (N3) group through a linker to the 5’ end of a DNA fragment,
`which is then used to couple with the triarylphosphine moiety on a solid surface. DMSO, dimethylsulfonyl oxide.
`
`Figure 6A-6B: Ligate the looped primer (B) to the immobilized single stranded DNA template forming a self primed
`DNA template moiety on a solid surface. P (in circle), phosphate.
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`Figure 7: Examples of structures of four nucleotide analogues for use in the sequencing by synthesis approach.
`Each nucleotide analogue has a unique fluorescent dye attached to the base through a photocleavable linker and
`the 3’-OH is either exposed or capped with a MOM group or an allyl group. FAM, 5-carboxyfluorescein; R6G, 6-
`carboxyrhodamine-6G; TAM, N,N,N’,N’-tetramethyl-6-carboxyrhodamine; ROX, 6-carboxy-X-rhodamine. R = H,
`CH2OCH3 (MOM) or CH2CH=CH2 (Allyl).
`
`Figure 8: A representative scheme for the