Long PCR for VNTR analysis.
The Polymerase Chain Reaction (PCR) has revolutionized the analysis of DNA from a variety of sources. With its sensitivity and ability to amplify degraded DNAs and small quantities of samples, coupled with fast turn-around-time, PCR is often the analytical method of choice for DNA profiling in forensic laboratories. RFLP methods, while requiring larger amounts of high molecular weight DNA and needing approximately 6-8 weeks of analytical time, still provide a higher power of discrimination per locus than that achieved using the loci currently available for PCR. The combination of both RFLP and PCR would be advantageous for some applications. A new technique, Long PCR, allows for the effective amplification of long DNA targets from approximately 0.5 kb to > 20 kb of genomic DNA. Currently, several Long PCR systems are commercially available. Using a Taq/Pyrococcus DNA polymerase enzyme system and DNA isolated from bloodstains, we have successfully amplified 1-20 ng of Chelex-extracted DNA, an amount commonly used in Amp-FLP technology. The robustness of Long PCR in comparison to RFLP was also examined through the use of partially degraded blood samples. Long PCR was then used to amplify both D2S44 and D5S110 RFLP loci. Although all D2 and D5 alleles were detected, the larger alleles were amplified at significantly lower levels than the smaller alleles.
Richie
K L
KL
Chemical Sciences and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA.
Goldsborough
M D
MD
Darfler
M M
MM
Benzinger
E A
EA
Lovekamp
M L
ML
Reeder
D J
DJ
O'Connell
C D
CD
D000595:Amino Acid Sequence; D001801:Blood Stains; D004247:DNA / Q000032:analysis; D016172:DNA Fingerprinting / Q000379:methods*; D005554:Forensic Medicine / Q000379:methods*; D006801:Humans; D018598:Minisatellite Repeats; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012016:Reference Values; D015203:Reproducibility of Results
D004247:DNA