Helicase suppression of non-template amplification

Patent metadata

Field	Value
Publication number	US-9920358-B2
Application number	US-201414385674-A
Country	US
Kind code	B2
Filing date	Jun 27, 2014
Priority date	Jun 27, 2013
Publication date	Mar 20, 2018
Grant date	Mar 20, 2018

Abstract

Provided herein is a method for reducing amplification of non-template molecules in a nucleic acid sample. In certain embodiments, the method involves adding a helicase to a reaction mixture for non-helicase-dependent amplification of target nucleic acid.

First claim

The invention claimed is:

1. A reaction mixture comprising:
a) a nucleic acid sample comprising a template;
b) nucleotides;
c) four or more primers;
d) a polymerase; and
e) a non-thermostable helicase;
wherein the reaction mixture does not contain a single-stranded DNA binding protein (SSBP) and wherein the reaction mixture is capable of amplifying the template when placed under isothermal or polymerase chain reaction conditions.

2. The reaction mixture of claim 1 , wherein the helicase is a PcrA/UvrD/Rep helicase.

3. The reaction mixture of claim 1 , wherein the polymerase is a strand-displacing polymerase.

4. The reaction mixture of claim 3 , wherein the polymerase is selected from the group consisting of a Bst polymerase, a polD polymerase, a 9° N polymerase and phi29 polymerase.

5. The reaction mixture of claim 1 , wherein the polymerase is a thermostable polymerase.

6. The reaction mixture of claim 1 , wherein the template is RNA and the polymerase is a reverse transcriptase.

7. The reaction mixture of claim 1 , wherein the template is genomic DNA.

8. A method for reducing amplification of non-template molecules from a nucleic acid sample, comprising:
a) incubating a reaction mixture of claim 1 under amplification conditions, and
b) amplifying the template; wherein the amplification reaction is not helicase dependent but wherein the helicase reduces amplification of non-template molecules.

9. The method of claim 8 , wherein the amplification conditions are isothermal amplification conditions.

10. The method of claim 8 , wherein (b) results in whole genome amplification.

11. The method of claim 8 , wherein (b) results in amplification of one or more target fragments of a genome or cDNA.

12. The method of claim 8 , wherein the amplification conditions comprise thermocycling.

13. The method of claim 8 , wherein (b) results in reverse transcription of an RNA template.

14. The method of claim 13 , wherein (b) results in amplification of an RNA template by RT-PCR.

15. The method of claim 8 , wherein the method further comprises quantifying the amount of amplified template after (b).

16. The method of claim 8 , wherein the helicase is a PcrA/UvrD/Rep helicase.

17. The method of claim 8 , wherein the polymerase is selected from the group consisting of a Bst polymerase, a polD polymerase, 9° N polymerase and phi29 polymerase.

18. The method of claim 8 , wherein the reaction mixture is a loop-mediated isothermal amplification (LAMP) reaction mixture.

19. A loop-mediated isothermal amplification (LAMP) reaction mixture comprising:
a) a nucleic acid sample comprising a template;
b) nucleotides;
c) four or more primers;
d) a polymerase; and
e) a helicase;
wherein the reaction mixture optionally contains a single-stranded DNA binding protein (SSBP) and wherein the reaction mixture is capable of amplifying the template by LAMP.

20. The LAMP reaction mixture of claim 19 , wherein the mixture comprises six or more primers.

Assignees

• New England Biolabs Inc

Inventors

• Tanner Nathan
• Evans Jr Thomas C

Classifications

• C12Q1/6848 Primary

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C12Q2527/101

  Temperature · CPC title

• C12Q2521/513

  Winding/unwinding enzyme, e.g. helicase · CPC title

• C12Q1/6853 Primary

  using modified primers or templates · CPC title