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 comprising:
a) subjecting a reaction mixture to from 25-40 two-step amplification cycles and thereby forming an amplicon mixture, the amplicon mixture comprising an amplicon of each of the following short tandem repeat loci: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA; and
b) separating the amplicon mixture by capillary electrophoresis, and thereby determining alleles present at D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA.
2. The method of claim 1 , where in the two-step amplification cycle comprises an annealing temperature from 55° C. to 75° C.
3. The method of claim 2 , wherein the two-step amplification cycle comprises an annealing temperature from 59° C. to 75° C.
4. The method of claim 1 , wherein the two-step amplification cycle comprises a denaturing temperature from 85° C. to 100° C.