Massive parallel method for decoding DNA and RNA

Patent metadata

Field	Value
Publication number	US-10662472-B2
Application number	US-201816200557-A
Country	US
Kind code	B2
Filing date	Nov 26, 2018
Priority date	Oct 6, 2000
Publication date	May 26, 2020
Grant date	May 26, 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 cytosine deoxyribonucleotide analogue for sequencing a DNA template by stepwise synthesis of a complementary, growing DNA strand using a DNA polymerase reaction and stepwise detection of nucleotide analogues incorporated into the growing DNA strand, 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 the DNA polymerase or with incorporation of the analogue into the growing DNA strand during the DNA polymerase reaction, (c) is stable during the 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 the DNA polymerase reaction;
wherein Tag represents a detectable fluorescent moiety;
wherein (i) the cytosine deoxyribonucleotide analogue is incorporated into the growing DNA strand as a result of the DNA polymerase reaction, (ii) the Tag is detected and thereby the incorporated analogue is detected, and (iii) the covalent bond between the 3′-oxygen and R is chemically cleaved under conditions enabling the growing DNA strand to survive the washing, detection and cleavage processes and to remain annealed to the DNA template so as to allow the incorporation and detection of the next nucleotide analogue;
wherein Y represents a chemically cleavable, chemical linker which (a) does not interfere with recognition of the analogue as a substrate by the DNA polymerase or with incorporation of the analogue into the growing DNA strand and (b) is stable during the DNA polymerase reaction; and
wherein the cytosine deoxyribonucleotide analogue:
i) is recognized as a substrate by the DNA polymerase for incorporation into the growing DNA strand,
ii) is efficiently and faithfully incorporated at the end of the growing DNA strand during the DNA polymerase reaction,
iii) produces a 3′—OH group on the deoxyribose upon cleavage of R under conditions compatible with DNA sequencing,
iv) no longer includes a tag on the base upon cleavage of Y under conditions compatible with DNA sequencing, and
v) is capable of forming hydrogen bonds with guanine or a guanine nucleotide analogue.

2. A cytosine deoxyribonucleotide analogue for sequencing a DNA template by stepwise synthesis of a complementary, growing DNA strand using a DNA polymerase reaction and stepwise detection of nucleotide analogues incorporated into the growing DNA strand, 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 the DNA polymerase or with incorporation of the analogue into the growing DNA strand during the DNA polymerase reaction, (c) is stable during the 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 the DNA polymerase reaction;
wherein Tag represents a detectable fluorescent moiety;
wherein (i) the cytosine deoxyribonucleotide analogue is incorporated into the growing DNA strand as a result of the DNA polymerase reaction, (ii) the Tag is detected and thereby the incorporated analogue is detected, and (iii) the covalent bond between the 3′-oxygen and R is chemically cleaved under conditions enabling the growing DNA strand to survive the washing, detection and cleavage processes and to remain annealed to the DNA template so as to allow the incorporation and detection of the next nucleotide analogue;
wherein Y represents a chemically cleavable, chemical linker which (a) does not interfere with recognition of the analogue as a substrate by the DNA polymerase or with incorporation of the analogue into the growing DNA strand and (b) is stable during the DNA polymerase reaction; and
wherein the cytosine deoxyribonucleotide analogue:
i) is recognized as a substrate by the DNA polymerase for incorporation into the growing DNA strand,
ii) is efficiently and faithfully incorporated at the end of the growing DNA strand during the DNA polymerase reaction,
iii) produces a 3′—OH group on the deoxyribose upon cleavage of R under conditions compatible with DNA sequencing,
iv) no longer includes a tag on the base upon cleavage of Y under conditions compatible with DNA sequencing, and
v) is capable of forming hydrogen bonds with guanine or a guanine nucleotide analogue.

Assignees

• Univ Columbia

Inventors

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

Classifications

• C12Q1/68

  involving nucleic acids · CPC title

• C07B2200/11

  Compounds covalently bound to a solid support · CPC title

• C12Q1/6874

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

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C40B40/00

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