Kristy L. Richie, Catherine D. O'Connell and Dennis J. Reeder
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD
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 0.5-20kb of genomic DNA. These size ranges are similar to those used in forensic laboratories performing RFLP analyses. Amplification of these RFLP target areas, if possible, would bring the speed, simplicity and sensitivity of PCR to RFLP-based technologies. In addition, it would allow the forensic community to utilize the well established RFLP data bank-CODIS.
Ideally, one would like to obtain fragments through Long PCR techniques that are identical to those produced by restriction endonucleases using RFLP analysis. However, very little information is available concerning the DNA sequence and restriction enzyme sites surrounding the RFLP probes. If such information cannot be obtained, we intend to isolate genomic DNA clones containing RFLP markers and sequence the regions surrounding the restriction enzyme sites. PCR primers will then be designed for amplification by Long PCR.
There are currently three Long PCR systems commercially available. We are examining these systems to determine their feasibility for use with forensic samples. The systems will be evaluated for robustness, minimum amount of forensic material necessary for amplification and ultimately, their ability to generate PCR products corresponding to the most commonly used RFLP markers.
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