Thiolated nucleotide analogues for nucleic acid synthesis

Patent metadata

Field	Value
Publication number	US-2016130644-A1
Application number	US-201514937211-A
Country	US
Kind code	A1
Filing date	Nov 10, 2015
Priority date	Nov 11, 2014
Publication date	May 12, 2016
Grant date	—

Abstract

The present disclosure provide systems, compositions, methods, reagents, kits and products for extending a nucleic acid that includes incorporating a nucleotide residue at a terminus of a nucleic acid using a polymerase enzyme and at least one nucleotide, wherein the at least one nucleotide includes a thiophosphate moiety, and wherein the at least one nucleotide is resistant to hydrolysis by phosphatase. In some embodiments, the nucleotide incorporation can be conducted in the presence of a phosphatase. In some embodiments, the nucleotide incorporation can be conducted in the presence of at least on chelation moiety that is configured to bind an orthophosphate moiety.

First claim

What is claimed:

1 . A method of identifying a base at a position in a target nucleic acid, comprising:
a) incorporating a nucleotide at a terminus of an extension primer that is hybridized to the target nucleic acid using a polymerase enzyme and at least one nucleotide, wherein the terminal phosphate of the at least one nucleotide includes a thio-phosphate moiety, wherein the at least one nucleotide is resistant to hydrolysis by a phosphatase enzyme, and wherein the nucleotide incorporation produces a thio-pyrophosphate and a hydrogen ion or a proton; and b) identifying the nucleotide that is incorporated at the terminus of the extension primer.

2 . The method of claim 1 , further comprising: identifying the nucleotide that is incorporated at the terminus of the extension primer by detecting the hydrogen ion or the proton that is produced upon incorporation of the nucleotide.

3 . The method of claim 1 , wherein the at least one nucleotide is resistant to hydrolysis by a pyrophosphatase enzyme.

4 . The method of claim 1 , further comprising: performing the nucleotide incorporating step in the presence of a phosphatase enzyme.

5 . The method of claim 1 , wherein the at least one nucleotide comprises a deoxyribonucleotide-5′-γ[gamma]-thio-triphosphate.

6 . The method of claim 3 , further comprising: performing the nucleotide incorporating step in the presence of a pyrophosphatase.

7 . The method of claim 4 , further comprising: hydrolyzing the thio-pyrophosphate in the presence of the phosphatase, thereby producing an orthophosphate.

8 . The method of claim 1 , further comprising: identifying the nucleotide that is incorporated at the terminus of the extension primer by detecting the hydrogen ion or the proton.

9 . The method of claim 7 , wherein the nucleotide incorporation step is conducted in the presence of at least one chelation moiety, wherein the chelation moiety is configured to bind the orthophosphate moiety.

10 . The method of claim 9 , wherein the orthophosphate is selected from a monobasic orthophosphate, a dibasic orthophosphate, a tribasic orthophosphate, a monobasic thiophosphate, a dibasic thiophosphate, and a tribasic thiophosphate.

11 . The method of claim 9 , further comprising binding the at least one chelation moiety to the orthophosphate.

12 . The method of claim 1 , wherein the specific rate of incorporation of the nucleotide with the thiophosphate moiety is at least 95% of the specific rate of incorporation of the analogous nucleotide without the thiophosphate moiety.

13 . The method of claim 1 , wherein the polymerase enzyme is a Bst polymerase.

14 . The method of claim 1 , wherein the polymerase enzyme is a mutant Bst polymerase.

15 . The method of claim 1 , wherein the incorporating the nucleotide is conducted in a reaction chamber that is operatively coupled at least one ion sensor that detects hydrogen ions or protons.

16 . The method of claim 15 , wherein the at least one ion sensor comprises and ISFET.

17 . The method of claim 1 , wherein the incorporating the nucleotide is conducted on an array of reaction chambers, wherein individual reaction chambers in the array are operatively coupled to at least one ion sensor that detects hydrogen ions or protons.

18 . The method of claim 17 , wherein individual reaction chambers in the array are operatively coupled to at least one ISFET.

Assignees

• Life Technologies Corp

Inventors

• Menchen Steven
• Nikiforov Theo

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• G01N27/4145

  specially adapted for biomolecules, e.g. gate electrode with immobilised receptors · CPC title