Compositions and methods for reducing background DNA amplification

Patent metadata

Field	Value
Publication number	US-9546358-B2
Application number	US-201313799463-A
Country	US
Kind code	B2
Filing date	Mar 13, 2013
Priority date	Jun 4, 2012
Publication date	Jan 17, 2017
Grant date	Jan 17, 2017

Abstract

Compositions are provided that include a plurality of small molecules selected from the group consisting of an amide, urea or acetone having a molecular weight less than 300 g/mol; and dNTPs and a polymerase in a buffer suitable for use as an amplification buffer. Methods of use of the compositions are also described for reducing non-template DNA amplification.

First claim

What is claimed is:

1. A method comprising:
(a) producing a reaction mixture by combining:
(i) a carboxamide selected from the group consisting of formamide, acetamide, propionamide, butyramide, isobutryamide, valeramide and hexanamide; and an N-alkylcarboxamide selected from the group consisting of N-methylformamide, N-methylacetamide, N,N-dimethylformamide, N,N-diethylformamide, N,N-dimethylbutyramide, at a molar ratio in the range of 1:1 to 1:10 (carboxymide:N-alkylcarboxamide),
(ii) a polymerase;
(iii) dNTPs;
(iv) one or more primers;
(v) template and non-template nucleic acid; and
(vi) a DNA amplification buffer; and

(b) incubating the reaction mixture under conditions to amplify the template nucleic acid, wherein the carboxamide and N-alkylcarboxamide synergistically reduce amplification of the non-template nucleic acid.

2. The method of claim 1 , wherein the carboxamide is at a concentration in the range of 0.02 M to 0.7 M and the N-alkyl carboxamide is at a concentration in the range of 0.03 M to 1.5 M.

3. The method of claim 1 , wherein the polymerase is a family A polymerase or a family B polymerase.

4. The method of claim 1 , wherein the polymerase is selected from the group consisting of Bst polymerase, PolD polymerase, KOD polymerase, mutants of the same and chimeras of the same.

5. The method of claim 1 , wherein the polymerase is a strand-displacing polymerase.

6. The method of claim 1 , wherein the conditions of step (b) are isothermal.

7. The method of claim 1 , wherein the conditions of step (b) comprise thermocycling.

8. The method of claim 1 , wherein the reaction mixture comprises isobutryamide and N-methylformamide.

Assignees

• New England Biolabs Inc

Inventors

• Tanner Nathan
• Evans 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

• C12N9/1241 Primary

  Nucleotidyltransferases (2.7.7) · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title