Methods of using nucleotide analogues

Patent metadata

Field	Value
Publication number	US-10273539-B2
Application number	US-201615343279-A
Country	US
Kind code	B2
Filing date	Nov 4, 2016
Priority date	Nov 6, 2015
Publication date	Apr 30, 2019
Grant date	Apr 30, 2019

Abstract

The present invention provides methods, compositions, mixtures and kits utilizing deoxynucleoside triphosphates comprising a 3′-O position capped by a group comprising methylenedisulfide as a cleavable protecting group and a detectable label reversibly connected to the nucleobase of said deoxynucleoside. Such compounds provide new possibilities for future sequencing technologies, including but not limited to Sequencing by Synthesis.

First claim

We claim:

1. A method of performing a DNA synthesis reaction comprising the steps of
a) providing a nucleic acid template with a primer hybridized to said template, a DNA polymerase, at least one deoxynucleoside triphosphate having the structure:

 wherein D is a cleavable protecting group selected from the group consisting of an disulfide alkyl, disulfide substituted alkyl groups, disulfide allyl, and disulfide substituted allyl groups; B is a nucleobase; Linker comprises a cleavable oxymethylenedisulfide-containing site core, wherein said cleavable site core is selected from the group consisting of:

 wherein R 1 and R 2 are independently selected alkyl groups; and Label is a detectable label selected from the group consisting of fluorophore dyes, energy transfer dyes, mass-tags, biotin, and haptenes, and
b) subjecting said reaction mixture to conditions which enable a DNA polymerase catalyzed primer extension reaction.

2. The method according to claim 1 , wherein said DNA polymerase catalyzed primer extension reaction is part of a sequencing reaction.

3. A method for analyzing a DNA sequence comprising the steps of
a) providing a nucleic acid template with a primer hybridized to said template forming a primer/template hybridization complex,
b) adding DNA polymerase, and a first deoxynucleoside triphosphate having the structure:

 wherein D is a cleavable protecting group selected from the group consisting of an disulfide alkyl, disulfide substituted alkyl groups, disulfide allyl, and disulfide substituted allyl groups; B is a nucleobase; Linker comprises a cleavable oxymethylenedisulfide-containing site core, wherein said cleavable site core is selected from the group consisting of:

 wherein R 1 and R 2 are independently selected alkyl groups; and Label is a detectable label selected from the group consisting of fluorophore dyes, energy transfer dyes, mass-tags, biotin, and haptenes,
c) subjecting said reaction mixture to conditions which enable a DNA polymerase catalyzed primer extension reaction so as to create a modified primer/template hybridization complex, and
d) detecting said first detectable label of said deoxynucleoside triphosphate in said modified primer/template hybridization complex.

4. The method according to claim 3 , further comprising the steps of e) removing said cleavable protecting group, and f) repeating steps b) to e) at least once.

5. The method according to claim 4 , wherein the method further comprises adding a second deoxynucleoside triphosphate during repeat of step b), wherein said second deoxynucleoside triphosphate comprises a second detectable label attached via a cleavable oxymethylenedisulfide linker, wherein said second detectible label is different from said first detectible label.

6. The method according to claim 5 , wherein the nucleobase of said second deoxynucleoside triphosphate is different from the nucleobase of said first deoxynucleoside triphosphate.

7. The method according to claim 4 , wherein a mixture of at least 4 differently labeled, 3′-O methylenedisulfide capped deoxynucleoside triphosphate compounds representing analogs of Adenosine, Guanosine, Cytidine, and Thymidine or Uridine are used in step b).

8. The method according to claim 4 , wherein step e) is performed by exposing said modified primer/template hybridization complex to a reducing agent.

9. The method according to claim 8 , wherein said reducing agent is tris(2-carboxyethyl)phosphine.

10. The method according to claim 4 , wherein step e) is performed by exposing said modified primer/template hybridization complex to a thiol-containing compound.

11. The method according to claim 3 , wherein said detecting allows for the determination of the nucleobase of said incorporated first deoxynucleoside triphosphate.

12. The method according to claim 4 , wherein said detectable label from said modified primer/template hybridization complex is removed prior to step e).

13. A method of performing a DNA synthesis reaction comprising the steps of
a) providing a nucleic acid template with a primer hybridized to said template, a DNA polymerase, at least one deoxynucleoside triphosphate having the structure:

 wherein D is a cleavable protecting group selected from the group consisting of an disulfide alkyl, disulfide substituted alkyl groups, disulfide allyl, and disulfide substituted allyl groups; B is a nucleobase; A is an attachment group selected from the group consisting of propargyl, exocyclic amine, propargyl amine, and propargyl hydroxyl; C is a cleavable site core selected from the group consisting of:

 wherein R 1 and R 2 are independently selected alkyl groups; L 1 and L 2 are connecting groups; and Label is a detectable label selected from the group consisting of fluorophore dyes, energy transfer dyes, mass-tags, biotin, and haptenes, and
b) subjecting said reaction mixture to conditions which enable a DNA polymerase catalyzed primer extension reaction.

14. The method according to claim 13 , wherein said DNA polymerase catalyzed primer extension reaction is part of a sequencing reaction.

15. The method according to claim 13 , wherein L 1 is selected from the group consisting of —CONH(CH 2 ) x —, —CO—O(CH 2 ) x —, —CONH—(OCH 2 CH 2 O) x —, —CO—O(CH 2 CH 2 O) x —, and —CO(CH 2 ) x —, wherein x is 0-10.

16. The method according to claim 13 , wherein L 2 is selected from the group consisting of

—NH—, —(CH 2 ) x —NH—, —C(Me) 2 (CH 2 ) x NH—, —CH(Me)(CH 2 ) x NH—, —C(Me) 2 (CH 2 ) x CO—, —CH(Me)(CH 2 ) x CO—, —(CH 2 ) x OCONH(CH 2 ) y O(CH 2 ) z NH—, —(CH 2 ) x CONH(CH 2 CH 2 O) y (CH 2 ) z NH—, —(CH 2 ) x OCONH(CH 2 CH 2 O) y (CH 2 ) z NH—, —CONH(CH 2 ) x —, and —CO(CH 2 ) x —, wherein x, y, and z are each independently selected from is 0-10.

17. A method for analyzing a DNA sequence comprising the steps of
a) providing a nucleic acid template with a primer hybridized to said template forming a primer/template hybridization complex,
b) adding DNA polymerase, and a first deoxynucleoside triphosphate having the structure:

 wherein D is a cleavable protecting group selected from the group consisting of an disulfide alkyl, disulfide substituted alkyl groups, disulfide allyl, and disulfide substituted allyl groups; B is a nucleobase; A is an attachment group selected from the group consisting of propargyl, exocyclic amine, propargyl amine, and propargyl hydroxyl; C is a cleavable site core selected from the group consisting of:

 wherein R 1 and R 2 are independently selected alkyl groups; L 1 and L 2 are connecting groups; and Label is a detectabl

Assignees

• Intelligent Biosystems Inc
• Qiagen Sciences Llc

Inventors

• Marma Mong Sano
• Olejnik Jerzy

Classifications

• C07D207/40

  2,5-Pyrrolidine-diones · CPC title

• C09B23/16

  the polymethine chain containing hetero atoms · CPC title

• C09B1/00

  Dyes with anthracene nucleus not condensed with any other ring · CPC title

• C07F7/1804

  Compounds having Si-O-C linkages (Si-O-acyl linkages C07F7/1896) · CPC title

• C09B23/083

  five >CH- groups · CPC title