Massive parallel method for decoding DNA and RNA

Patent metadata

Field	Value
Publication number	US-9868985-B2
Application number	US-201715647657-A
Country	US
Kind code	B2
Filing date	Jul 12, 2017
Priority date	Oct 6, 2000
Publication date	Jan 16, 2018
Grant date	Jan 16, 2018

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 analog after the nucleotide analog is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogs which comprise unique labels attached to the nucleotide analog 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 sequencing a nucleic acid which comprises detecting the identity of a nucleotide analogue incorporated into the end of a growing strand of DNA in a polymerase reaction, wherein the nucleotide analogue is any of the following:

wherein R (a) represents a small, chemically cleavable, chemical group capping the oxygen at the 3′ position of the deoxyribose of tl 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 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;
wherein the nucleotide 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.

2. A method for simultaneously sequencing a plurality of different nucleic acids which comprises simultaneously applying the method of claim 1 to the plurality of different nucleic acids.

Assignees

• Univ Columbia

Inventors

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

Classifications

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C40B40/00

  Libraries per se, e.g. arrays, mixtures · CPC title

• C12Q2535/101

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

• C07B2200/11

  Compounds covalently bound to a solid support · CPC title

• C12Q2525/117

  incorporating modified base · CPC title