Heat-soaked PCR: an efficient method for DNA amplification with applications to forensic analysis.
Heat-soaked PCR (HS-PCR) is a method for enhancing amplification performed by heating the DNA sample at 94 degrees C in 90 microliters 1.1 x buffer for 30 min. A 10-microliters bolus of concentrated (10x) deoxynucleotides, Taq DNA polymerase and primers prepared without buffer is then added just prior to thermal cycling. We have investigated the application of this method in a variety of forensically important DNA samples and compared it with regular PCR (R-PCR). DNA samples extracted from bone, postmortem tissues, bloodstains and hair contained low concentrations of human DNA or were contaminated with either non- human DNA or hemoglobin degradation products. Optimal conditions for HS-PCR were determined for the 3' ApoB VNTR locus and applied to a centromeric repeat element and to a single-copy locus. HS-PCR consistently and reproducibly enhanced product yield and specificity over R-PCR at all three loci in the entire set of DNA samples. HS-PCR was also effective in overcoming the inhibitory effect of hemoglobin at concentrations that fully impeded R-PCR.
Ruano
G
G
Forensic Science Laboratory, Connecticut State Police, Meriden 06450.
Pagliaro
E M
EM
Schwartz
T R
TR
Lamy
K
K
Messina
D
D
Gaensslen
R E
RE
Lee
H C
HC
D004247:DNA / Q000032:analysis* / Q000097:blood; D005554:Forensic Medicine / Q000379:methods*; D006454:Hemoglobins; D006358:Hot Temperature; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011180:Postmortem Changes
D006454:Hemoglobins; D004247:DNA