Kristy L. Richie¹, Dennis J. Reeder¹, Mindy D. Goldsborough², Marlene M. Darfler² and Catherine D. O’Connell^1
1 Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD
² Life Technologies, Inc., Gaithersburg, MD

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In recent years, the Polymerase Chain Reaction (PCR) has revolutionized the approach to the recovery of DNA from a variety of sources. With its extreme sensitivity and its ability to amplify degraded DNAs and small quantities of samples, coupled with its fast turn-around time, PCR is often the analytical method of choice for DNA profiling in many forensic laboratories. Nonetheless, many laboratories still use the more traditional RFLP approach to DNA profiling. RFLP methods, while requiring large amounts of high molecular weight DNA and needing approximately 6-8 weeks of analytical time, still provide a higher power of discrimination due to the availability of many highly polymorphic marker systems compared to PCR-based systems. Moreover, RFLP-based systems have found general acceptance in most courts. That, coupled with the existence of a well established RFLP data bank–CODIS–makes the RFLP method of analysis extremely robust.

The combining of these two techniques might prove to be advantageous for some applications. Recently, a new approach to PCR amplification has been developed, allowing one to amplify larger fragments of template DNA. The technique, Long PCR, generally uses a unique enzyme mix optimized for both polymerase and proofreading activities, allowing for the effective amplification of long DNA targets from approximately 0.5 kb to > 20 kb of genomic DNA. There are currently a number of Long PCR systems commercially available. We have examined three of these systems to determine their feasibility for use with forensic samples. From initial observations, we have determined that the enzyme system using Taq/Pyrococcus DNA polymerase mix most consistently amplified the tPA gene-specific 9.3 kb and 4.8 kb DNA regions examined. Using this system, we have successfully amplified DNA extracted from 1/20 of an 1/8" blood stain, an amount commonly used in Amp-FLP technology. PCR products were detectable after amplification of the long, single copy tPA gene regions as well as the short tandem repeat units within the D1S80 locus.

Our current studies focus on the length of product one can amplify from blood using Long PCR, and the robustness of Long PCR in comparison to RFLP when the blood samples are partially degraded. We are also using Long PCR to amplify the D5 RFLP locus. Ultimately, we hope to achieve the ability to generate PCR products corresponding to all of the most commonly used RFLP markers.