Massive parallel method for decoding DNA and RNA

Patent metadata

Field	Value
Publication number	US-10669577-B2
Application number	US-201514670748-A
Country	US
Kind code	B2
Filing date	Mar 27, 2015
Priority date	Oct 6, 2000
Publication date	Jun 2, 2020
Grant date	Jun 2, 2020

Abstract

This invention provides methods for attaching a nucleic acid to a solid surface and for sequencing nucleic acid by detecting the identity of each nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogues which comprise unique labels attached to the nucleotide analogue through a cleavable linker, and a cleavable chemical group to cap the —OH group at the 3′-position of the deoxyribose.

First claim

What is claimed is:

1. A method for simultaneously sequencing different deoxyribonucleic acid templates immobilized on a solid surface, which comprises (1) simultaneously applying to said immobilized different deoxyribonucleic acid templates a sequencing by synthesis method comprising multiple cycles, and (2) detecting the identity of the nucleotide analogue incorporated onto each growing DNA strand hybridized to each of said different deoxyribonucleic acid templates, each cycle comprises the following steps:
(a) providing to the different deoxyribonucleic acid templates at least one nucleotide analogue having the structure:

wherein base is a nucleobase selected from the group consisting of A, C, G, T, and U, and derivatives thereof,
wherein R (a) represents a small, chemically cleavable chemical capping group, (b) does not interfere with recognition of the analogue as a substrate by a DNA polymerase, (c) is stable during a DNA polymerase reaction, and (d) does not contain a ketone group;
wherein OR is not a methoxy group, an ester group or an allyl ether group;
wherein the covalent bond between the 3′-oxygen and R is stable during a DNA polymerase reaction;
wherein Tag represents a detectable fluorescent moiety;
wherein Y represents a chemically cleavable, chemical linker which (a) does not interfere with recognition of the analogue as a substrate by a DNA polymerase and (b) is stable during a DNA polymerase reaction; and
wherein the analogue:
i) is recognized as a substrate by a DNA polymerase,
ii) is incorporated at the end of a growing strand of DNA during a DNA polymerase reaction,
iii) produces a 3′-OH group on the deoxyribose upon cleavage of R, and
iv) no longer includes a tag on the base upon cleavage of Y,
under conditions such that each growing strand is extended by incorporation of one nucleotide analogue per strand so as to create an extended growing DNA strand using a DNA polymerase reaction, said incorporated analogue serving as a reversible terminator of the polymerase reaction;

(b) detecting said detectable fluorescent moiety of said incorporated nucleotide analogue, so as to thereby identify the nucleotide analogue incorporated onto the growing strand; and
(c) removing the detectable fluorescent moiety and removing by chemical means the small, chemically cleavable chemical capping group of said incorporated analogue;
wherein a plurality of spots are on the solid surface, wherein each spot comprises a plurality of deoxyribonucleic acid templates having the same sequence;
wherein a plurality of deoxyribonucleic acid templates in a spot differs in sequence from a plurality of deoxyribonucleic acid templates in another spot; and
wherein a fluorescence emission from the detectable fluorescent moiety in each spot reveals the identity of the nucleotide analogue incorporated onto each growing DNA strand hybridized to the deoxyribonucleic acid templates in each spot.

2. The method of claim 1 wherein the at least one nucleotide analogue provided in step (a) is a 7-deaza guanine deoxyribonucleotide analogue having the structure:

3. The method of claim 1 wherein the at least one nucleotide analogue provided in step (a) is a 7-deaza adenine deoxyribonucleotide analogue having the structure:

4. The method of claim 1 wherein the at least one nucleotide analogue provided in step (a) is a cytosine deoxyribonucleotide analogue having the structure:

5. The method of claim 1 wherein the at least one nucleotide analogue provided in step (a) is a thymine deoxyribonucleotide analogue having the structure:

6. The method of claim 1 , wherein said plurality of deoxyribonucleic acid templates is able to self-prime initiation of the DNA polymerase reaction in step (a).

7. The method of claim 1 , wherein the deoxyribonucleic acid templates having the same sequence are generated by a polymerase chain reaction.

8. A method for sequencing a nucleic acid which comprises detecting the identity of a nucleotide analogue incorporated onto the end of a growing DNA strand in a polymerase reaction, wherein the nucleotide analogue is any of the following:

wherein R (a) represents a small, chemically cleavable chemical capping group, (b) does not interfere with recognition of the analogue as a substrate by a DNA polymerase, (c) is stable during a DNA polymerase reaction, and (d) does not contain a ketone group;
wherein OR is not a methoxy group, an ester group or an allyl ether group;
wherein the covalent bond between the 3′-oxygen and R is stable during a DNA polymerase reaction;
wherein Tag represents a detectable fluorescent moiety;
wherein Y represents a chemically cleavable, chemical linker which (a) does not interfere with recognition of the analogue as a substrate by a DNA polymerase and (b) is stable during a DNA polymerase reaction; and
wherein the analogue:
i) is recognized as a substrate by a DNA polymerase;
ii) is incorporated at the end of a growing strand of DNA during a DNA polymerase reaction;
iii) produces a 3′-OH group on the deoxyribose upon cleavage of R; and
iv) no longer includes a tag on the base upon cleavage of Y; and
wherein if the nucleotide analogue is: (A), it is capable of forming hydrogen bonds with cytosine or a cytosine nucleotide analogue; (B), it is capable of forming hydrogen bonds with thymine or a thymine nucleotide analogue; (C), it is capable of forming hydrogen bonds with guanine or a guanine nucleotide analogue; or (D), it is capable of forming hydrogen bonds with adenine or an adenine nucleotide analogue;
wherein in the method a plurality of nucleic acid templates having the same sequence as the nucleic acid is immobilized in a spot on a solid surface, wherein the spot comprises a plurality of growing DNA strands hybridized to the plurality of nucleic acid templates, and wherein a plurality of nucleotide analogues having the same detectable fluorescent moiety is incorporated onto the end of the plurality of growing DNA strands in a polymerase reaction, to thereby reveal the identity of the incorporated nucleotide analogue hybridized to the plurality of nucleic acid templates in the spot.

9. The method of claim 8 , wherein the plurality of nucleic acid templates having the same sequence is generated by a polymerase chain reaction.

10. A method for simultaneously sequencing a plurality of different nucleic acids which comprises simultaneously applying the method of claim 8 to each nucleic acid of the plurality of different nucleic acids, wherein a plurality of nucleic acid templates corresponding to each nucleic acid of the plurality of different nucleic acids is immobilized in a different spot on the solid surface.

11. A method for simultaneously sequencing a plurality of different nucleic acids which comprises simultaneously applying the method of claim 9 to each nucleic acid of the pl

Assignees

• Univ Columbia

Inventors

• Ju Jingyue
• Li Zengmin
• Edwards John Robert
• Itagaki Yasuhiro

Classifications

• C12Q2525/117

  incorporating modified base · CPC title

• C12Q2535/101

  Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators · CPC title

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C07B2200/11

  Compounds covalently bound to a solid support · CPC title