ISSN: 0022-1198
CODEN: JFSCA
Page Count: 10

Long PCR for VNTR Analysis
O'Connell, CD
Research Chemist, Biotechnology Division MS 8311 Chemical Sciences and Technology Laboratory, National Institute of Standards and Technology, MD

Reeder, DJ
Research Chemist, Biotechnology Division MS 8311 Chemical Sciences and Technology Laboratory, National Institute of Standards and Technology, MD

Benzinger, EA
Ohio Bureau of Criminal Identification and Investigation, OH

Richie, KL
Research Chemist, Biotechnology Division MS 8311 Chemical Sciences and Technology Laboratory, National Institute of Standards and Technology, MD

Goldsborough, MD
Research & Development Division, Life Technologies, Inc., MD

Lovekamp, ML
New Mexico Department of Public Safety Crime Laboratory, NM

Darfler, MM
Research & Development Division, Life Technologies, Inc., MD

(Received 24 July 1998; accepted 5 February 1999)

Abstract

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.

Keywords:
forensic science, RFLP, DNA typing, Long polymerase chain reaction

Paper ID: JFS14585J
DOI: 10.1520/JFS14585J
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