Helicase suppression of non-template amplification
View Patent ↗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.
1. A strand displacement amplification (SDA) reaction mixture comprising:
(a) nucleotides;
(b) a sequence-specific endonuclease having nicking activity under SDA conditions;
(c) a strand displacing polymerase; and
(d) a helicase;
wherein the reaction mixture does not contain a single-stranded DNA binding protein (SSBP) and wherein the reaction mixture is capable of amplifying a template DNA when placed under isothermal conditions.
2. The reaction mixture of claim 1 , wherein the helicase is a thermostable helicase.
3. The reaction mixture of claim 1 , wherein the helicase is a PcrA/UvrD/Rep helicase.
4. The reaction mixture of claim 1 , wherein the helicase is selected from the group consisting of a Thermoanaerobacter tengcongensis (Tte) helicase, Thermus thermophilus (Tth) helicase and Aquifex aeolicus (Aq793) helicase.
5. The reaction mixture of claim 1 , wherein the polymerase is selected from the group consisting of a Bst polymerase, a polD polymerase, a 9° N polymerase and phi29 polymerase.
6. The reaction mixture of claim 1 , wherein the template DNA is genomic DNA.
7. A method for reducing amplification of non-template DNA from a nucleic acid sample, comprising:
(a) incubating a reaction mixture of claim 1 with a template DNA under SDA amplification conditions; and
(b) amplifying the template DNA; wherein the amplification reaction is not helicase dependent but wherein the helicase reduces amplification of non-template DNA.
8. The method of claim 7 , wherein (b) results in whole genome amplification.
9. The method of claim 7 , wherein (b) results in amplification of one or more target fragments of a genome or cDNA.
10. The method according to claim 7 , wherein the template DNA is from a pathogenic entity.
11. The method of claim 7 , wherein the template DNA is bacterial or viral DNA.
12. The method of claim 7 , wherein the method further comprises quantifying the amount of amplified template after (b).
13. The method of claim 7 , wherein the helicase is a PcrA/UvrD/Rep helicase.
14. The method according to claim 7 , wherein the helicase is selected from the group consisting of a Thermoanaerobacter tengcongensis (Tte) helicase, Thermus thermophilus (Tth) helicase and Aquifex aeolicus (Aq793) helicase.
15. The method of claim 7 , wherein the polymerase is selected from the group consisting of a Bst polymerase, a polD polymerase, 9° N polymerase and phi29 polymerase.