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 kit for detection of an STR profile comprising:
a pair of primers configured to amplify at least one short tandem repeat (STR) locus;
a master mix composition for forming a reaction mixture with a sample;
an indicator probe; and
a Family A DNA polymerase provided so as to be present at a concentration of greater than 0.2 units/μl and up to 0.6 units/μl within the reaction mixture when formed.
2 . The kit of claim 1 , wherein the Family A polymerase comprises a polymerase from Thermus aquaticus, Thermus thermophilus HB-8, Thermus thermophilus HB-27, Thermus flavus, Thermus maritime ; large fragment, Thermotoga naepolitana, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus aggregans , or Thermomicrobium roseum.
3 . The kit of claim 2 , wherein the Family A polymerase comprises Taq DNA polymerase.
4 . The kit of claim 1 , wherein the Family A polymerase is antibody-bound.
5 . The kit of claim 1 , wherein the pair of primers is configured to amplify a region of the amelogenin locus.
6 . The kit of claim 1 , wherein the indicator probe is included in the master mix composition.
7 . The kit of claim 1 , wherein the indicator probe is a 5′-nuclease probe comprising a signal moiety capable of producing a detectable signal upon cleavage of the indicator probe.
8 . The kit of claim 1 , wherein the Family A DNA polymerase is provided so as to be present at a concentration of 0.3 units/μl to 0.6 units/μl within the reaction mixture when formed.
9 . The kit of claim 8 , wherein the Family A DNA polymerase is provided so as to be present at a concentration of 0.3 units/μl to 0.5 units/μl within the reaction mixture when formed.
10 . The kit of claim 1 , further comprising an indicator molecule configured to indicate an amount of nucleic acid in the reaction mixture.
11 . The kit of claim 1 , wherein the indicator probe is a is a hairpin probe comprising a signal moiety capable of producing a detectable signal.
12 . The kit of claim 1 , wherein the master mix composition comprises a divalent cation.
13 . The kit of claim 1 , further comprising an allelic ladder.
14 . The kit of claim 1 , further comprising an electrophoresis loading solution.
15 . The kit of claim 1 , wherein the master mix composition comprises deoxyribonucleotide triphosphates (dNTPs).
16 . The kit of claim 1 , wherein the kit comprises multiple pairs of primers each configured to amplify a different STR locus.
17 . The kit of claim 16 , wherein each primer is included so as to be present at a concentration of 0.1 μM or more within the reaction mixture when formed.
18 . A kit for detection of an STR profile comprising:
multiple pairs of primers each configured to amplify a different short tandem repeat (STR) locus;
a master mix composition for forming a reaction mixture with a sample;
an indicator probe comprising a signal moiety capable of producing a detectable signal upon cleavage of the indicator probe; and
an antibody-bund Taq polymerase provided so as to be present at a concentration of greater than 0.2 units/μl and up to 0.6 units/μl within the reaction mixture when formed.
19 . The kit of claim 18 , wherein the Taq polymerase is provided so as to be present at a concentration of 0.3 units/μl to 0.6 units/μl within the reaction mixture when formed.
20 . A method of amplifying a nucleic acid sequence, comprising:
subjecting a reaction mixture to at least one amplification cycle, wherein the reaction mixture comprises
a nucleic acid,
a pair of primers configured to amplify at least one short tandem repeat (STR), and
a Family A DNA polymerase included at a concentration of 0.3 units/μl to 0.6 units/μl;
wherein the time to complete one amplification cycle is 25 seconds or less.