Compositions and methods for reducing background DNA amplification
View Patent ↗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.
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.