Polyphenolic Additives in Sequencing by Synthesis

Patent metadata

Field	Value
Publication number	US-2019330690-A1
Application number	US-201916422056-A
Country	US
Kind code	A1
Filing date	May 24, 2019
Priority date	Feb 11, 2016
Publication date	Oct 31, 2019
Grant date	—

Abstract

The invention relates to methods, compositions, devices, systems and kits as described including, without limitation, reagents and mixtures for determining the identity of nucleic acids in nucleotide sequences using, for example, sequencing by synthesis methods. In particular, the present invention contemplates the use of polyphenolic compounds, known as antioxidant additives, to improve the efficiency of Sequencing-By-Synthesis reactions. For example, gallic acid (GA) is shown herein to be one of many exemplary SBS polyphenolic additives.

First claim

1 - 14 . (canceled)

15 . A method of incorporating labeled nucleotides, comprising:
a) providing
i) a plurality of nucleic acid primers and template molecules,
ii) a polymerase,
iii) a cleave reagent comprising a reducing agent and the polyphenolic compound gentisic acid, and
iv) a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable disulfide linker to the base;
b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers; c) incorporating a first labeled nucleotide analogue with said polymerase into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated labeled nucleotide analogue; d) detecting said incorporated labeled nucleotide analogue; and e) cleaving the cleavable linker of said incorporated nucleotide analogues with said cleave reagent.

16 . The method of claim 15 , wherein said reducing agent of said cleave reagent comprises TCEP (tris(2-carboxyethyl)phosphine).

17 . The method of claim 15 , wherein said incorporated nucleotide analogues of step c) further comprise a removable chemical moiety capping the 3′-OH group.

18 . The method of claim 16 , wherein the cleaving of step e) removes the removable chemical moiety capping the 3′-OH group.

19 . The method of claim 18 , wherein the method further comprises:
f) incorporating a second nucleotide analogue with said polymerase into at least a portion of said extended primers.

20 . The method of claim 15 , wherein said label is fluorescent.

21 . A method of incorporating labeled nucleotides, comprising:
a) providing
i) a plurality of nucleic acid primers and template molecules,
ii) a polymerase,
iii) a cleave reagent comprising a reducing agent and the polyphenolic compound pryocatechol, and
iv) a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable disulfide linker to the base;
b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers; c) incorporating a first labeled nucleotide analogue with said polymerase into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated labeled nucleotide analogue; d) detecting said incorporated labeled nucleotide analogue; and e) cleaving the cleavable linker of said incorporated nucleotide analogues with said cleave reagent.

22 . The method of claim 21 , wherein said reducing agent of said cleave reagent comprises TCEP (tris(2-carboxyethyl)phosphine).

23 . The method of claim 21 , wherein said incorporated nucleotide analogues of step c) further comprise a removable chemical moiety capping the 3′-OH group.

24 . The method of claim 22 , wherein the cleaving of step e) removes the removable chemical moiety capping the 3′-OH group.

25 . The method of claim 24 , wherein the method further comprises:
f) incorporating a second nucleotide analogue with said polymerase into at least a portion of said extended primers.

26 . The method of claim 21 , wherein said label is fluorescent.

27 . A method of incorporating labeled nucleotides, comprising:
a) providing
i) a plurality of nucleic acid primers and template molecules,
ii) a polymerase,
iii) a cleave reagent comprising a reducing agent and the polyphenolic compound pyrogallol, and
iv) a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable disulfide linker to the base;
b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers; c) incorporating a first labeled nucleotide analogue with said polymerase into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated labeled nucleotide analogue; d) detecting said incorporated labeled nucleotide analogue; and e) cleaving the cleavable linker of said incorporated nucleotide analogues with said cleave reagent.

28 . The method of claim 27 , wherein said reducing agent of said cleave reagent comprises TCEP (tris(2-carboxyethyl)phosphine).

29 . The method of claim 27 , wherein said incorporated nucleotide analogues of step c) further comprise a removable chemical moiety capping the 3′-OH group.

30 . The method of claim 28 , wherein the cleaving of step e) removes the removable chemical moiety capping the 3′-OH group.

31 . The method of claim 30 , wherein the method further comprises:
f) incorporating a second nucleotide analogue with said polymerase into at least a portion of said extended primers.

32 . The method of claim 27 , wherein said label is fluorescent.

Assignees

• Qiagen Sciences Llc

Inventors

• Olejnik Jerzy
• Perbost Michel Georges

Classifications

• C12Q1/6823 Primary

  Release of bound markers · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title