Polymerase inhibitors and related compositions and methods
The present disclosure includes compositions and methods for improved DNA amplification reactions. In particular, the present disclosure provides compositions and methods for hot-start PCR applications using DNA polymerase inhibitors that minimize non-specific DNA amplification by inactivating DNA polymerase at lower temperatures.
1. A method of amplifying a nucleic acid comprising:
(a) adding amplification reagents and a nucleic acid template to a DNA polymerase inhibited by a compound of formula (II):
or a salt thereof,
wherein:
A is selected from a monocyclic or bicyclic aryl, heteroaryl, or heterocyclyl group, each of which may be optionally substituted with 1, 2, or 3 substituents;
R 1 is C 6 -C 20 alkyl;
R 2 is selected from hydrogen and —COOH;
n is 1 or 2; and
R 3 is selected from —COOH and —SO 3 X, wherein X is selected from hydrogen, an alkali metal cation, and an ammonium cation;
(b) heating to a temperature of at least 80° C. to activate the DNA polymerase; and
(c) running through a thermal cycling protocol of appropriate times and temperatures for the amplification reagents and a nucleic acid template.
2. The method of claim 1 , wherein the amplification reagents comprise: deoxynucleotide triphosphates, buffer, a magnesium salt, and an oligonucleotide primer.
3. The method of claim 2 , wherein the amplification reagents comprise forward and reverse primers for a target on the nucleic acid template.
4. The method of claim 1 , wherein the DNA polymerase is activated by heating to a temperature above 90° C.
5. The method of claim 1 , wherein the thermal cycling protocol comprises a three temperature cycle of (i) a high temperature denaturation step, (ii) a low temperature annealing step, and (iii) a middle temperature extension step, repeated 5 or more times in succession.
6. The method of claim 5 , wherein the denaturation, annealing, and extension steps are repeated 20 or more times in succession.
7. The method of claim 1 , wherein the thermal cycling protocol comprises a two temperature cycle of a high temperature denaturation step, a middle/low temperature annealing/extension step repeated 5 or more times in succession.
8. The method of claim 7 , wherein the denaturation and annealing/extension steps are repeated 20 or more times in succession.
9. The method of claim 1 , wherein A is phenyl that is unsubstituted or substituted with 1, 2, 3, or 4 substituents independently selected from C 1 -C 4 alkyl, C 1 -C 4 alkoxy, and halo.
10. The method of claim 1 , wherein A is a 5- or 6-membered monocyclic heteroaryl that is unsubstituted or substituted with 1, 2, or 3 substituents independently selected from C 1 -C 4 alkyl, C 1 -C 4 alkoxy, and halo.
11. The method of claim 1 , wherein A is a bicyclic heterocyclyl group that is unsubstituted.
12. The method of claim 1 , wherein R 1 is C 8 -C 14 alkyl.
13. The method of claim 12 , wherein R 1 is C 12 alkyl.
14. The method of claim 1 , wherein R 2 is hydrogen.
15. The method of claim 1 wherein R 2 is-COOH.
16. The method of claim 1 , wherein n is 1.
17. The method of claim 1 , wherein n is 2.
18. The method of claim 1 , or a salt thereof, wherein R 3 is —COOH.
19. The method of claim 1 , or a salt thereof, wherein R 3 is —SO 3 X, wherein X is a sodium cation.
20. The method of claim 1 , wherein the compound of formula (II) is selected from: