Fast PCR for STR genotyping
Disclosed is a method of amplifying a nucleic acid sequence, wherein the method comprises subjecting a reaction mixture to at least one amplification cycle, wherein the reaction mixture comprises a double-stranded nucleic acid and at least two primers capable of annealing to complementary strands of the double-stranded nucleic acid and amplifying at least one short tandem repeat (STR) using a Family A DNA polymerase in a Fast PCR protocol having a two-step amplification cycle in 25 seconds or less. Also disclosed are real-time PCR methods using the two-step protocol and kits for STR profiling using the Fast PCR protocol.
1. A method of amplifying a nucleic acid sequence, wherein the method comprises:
subjecting a reaction mixture to at least one amplification cycle, wherein the reaction mixture comprises
a double-stranded nucleic acid,
at least two primers capable of annealing to complementary strands of the double-stranded nucleic acid and amplifying at least one short tandem repeat (STR), and
an antibody-bound Taq DNA polymerase included at a concentration of 0.3 units/μl to 0.4 units/μ 1 ,
wherein the at least one amplification cycle comprises
denaturing the double-stranded nucleic acid in the reaction mixture, and
annealing the at least two primers to complementary strands of the denatured double-stranded nucleic acid and extending the at least two primers to thereby amplify the at least one STR,
wherein the time to complete one amplification cycle is 25 seconds or less.
2. The method of claim 1 , wherein annealing the at least two primers to complementary strands of the denatured double-stranded nucleic acid and extending the at least two primers are performed at the same temperature.
3. The method of claim 1 , further comprising amplifying a region of an amelogenin locus.
4. The method of claim 1 , wherein the reaction mixture further comprises an indicator molecule that indicates the amount of nucleic acid in the reaction mixture.
5. The method of claim 1 , wherein the reaction mixture further comprises an indicator probe capable of selectively hybridizing to a strand of the double-stranded nucleic acid.
6. The method of claim 5 , wherein the indicator probe is a 5′-nuclease probe comprising a signal moiety capable of producing a detectable signal, and wherein extension of at least one of the at least two primers results in cleavage of the 5′ nuclease probe.
7. The method of claim 6 , wherein cleavage of the 5′-nuclease probe increases the detectable signal from the signal moiety.
8. The method of claim 1 , wherein the indicator probe comprises a hybridization-dependent probe.
9. The method of claim 8 , wherein the hybridization-dependent probe is a hairpin probe comprising a signal moiety capable of producing a detectable signal.
10. The method of claim 1 , wherein the number of amplified molecules produced in at least one of the at least one amplification cycle is from 1.6-fold to 2-fold the number of molecules present at the start of the at least one of the at least one amplification cycle.
11. The method of claim 1 , wherein the amplification efficiency of polymerase in at least one of the at least one amplification cycle is from 0.8 to 1.0.
12. The method of claim 1 , wherein denaturing the double-stranded nucleic acid is performed at a temperature from about 85° C. to 95° C.
13. A method of amplifying a nucleic acid sequence, wherein the method comprises:
subjecting a reaction mixture to at least one amplification cycle, wherein the reaction mixture comprises
a double-stranded nucleic acid,
at least two primers capable of annealing to complementary strands of the double-stranded nucleic acid and amplifying at least one short tandem repeat (STR), and
an antibody-bound Taq DNA polymerase included at a concentration of 0.3 units/μl to 0.5 units/μl,
wherein the at least one amplification cycle comprises
denaturing the double-stranded nucleic acid in the reaction mixture, and
annealing the at least two primers to complementary strands of the denatured double-stranded nucleic acid and extending the at least two primers to thereby amplify the at least one STR,
wherein the time to complete one amplification cycle is 25 seconds or less.
14. The method of claim 13 , wherein annealing the at least two primers to complementary strands of the denatured double-stranded nucleic acid and extending the at least two primers are performed at the same temperature.
15. The method of claim 13 , wherein the reaction mixture further comprises an indicator probe capable of selectively hybridizing to a strand of the double-stranded nucleic acid.
16. The method of claim 15 , wherein the indicator probe is a 5′-nuclease probe comprising a signal moiety capable of producing a detectable signal, and wherein extension of at least one of the at least two primers results in cleavage of the 5′ nuclease probe.
17. the method of claim 15 , wherein the indicator probe is a hairpin probe comprising a signal moiety capable of producing a detectable signal.
18. The method of claim 13 , wherein the number of amplified molecules produced in at least one of the at least one amplification cycle is from 1.6-fold to 2-fold the number of molecules present at the start of the at least one of the at least one amplification cycle.
19. The method of claim 13 , wherein the amplification efficiency of polymerase in at least one of the at least one amplification cycle is from 0.8 to 1.0.
20. The method of claim 13 , wherein denaturing the double-stranded nucleic acid is performed at a temperature from about 85° C. to 95° C.