Massive parallel method for decoding DNA and RNA

Patent metadata

Field	Value
Publication number	US-10435742-B2
Application number	US-201816150185-A
Country	US
Kind code	B2
Filing date	Oct 2, 2018
Priority date	Oct 6, 2000
Publication date	Oct 8, 2019
Grant date	Oct 8, 2019

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 thymine deoxyribonucleotide analogue having the structure:

wherein R (a) represents a small, chemically cleavable, chemical group capping the oxygen at the 3′ position of the deoxyribose of the deoxyribonucleotide analogue, (b) does not interfere with recognition of the analogue as a substrate by a DNA polymerase, (c) is stable during a DNA polymerase reaction, (d) does not contain a ketone group, and (e) is not a —CH 2 CH═CH 2 group;
wherein OR is not a methoxy group or an ester 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 thymine deoxyribonucleotide 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,
iv) no longer includes a tag on the base upon cleavage of Y, and
v) is capable of forming hydrogen bonds with adenine or an adenine nucleotide analogue.

2. A thymine deoxyribonucleotide analogue having the structure:

wherein R (a) represents a small, chemically cleavable, chemical group capping the oxygen at the 3′ position of the deoxyribose of the deoxyribonucleotide analogue, (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 thymine deoxyribonucleotide 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,
iv) no longer includes a tag on the base upon cleavage of Y, and
v) is capable of forming hydrogen bonds with adenine or an adenine nucleotide analogue.

Assignees

• Univ Columbia

Inventors

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

Classifications

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C07B2200/11

  Compounds covalently bound to a solid support · CPC title

• C07H21/00

  Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids · CPC title

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title