Deborah L. Fisher1, Barbara W. Koons2, Jenifer A. Lindsey1
and Bruce Budowle2
1DNA Unit, FBI Laboratory, 10th and Pennsylvania Ave, Washington DC
2Forensic Science Research and Training Center, FBI Academy, Quantico, VA
The reverse dot blot polymerase chain reaction (PCR)-based typing systems have proven to be beneficial to the forensic science community by providing information from minute quantities of potentially degraded DNA in a rapid manner. Extensive validation studies of the AmpliType™ PM kit have shown that reliable results can be obtained from human fluid/tissue samples subjected to a variety of substrates, environmental conditions, contamination, etc. Recently, problems have arisen at the GC locus when the procedure is employed outside the bounds of a validated protocol. Specifically, an elapsed time period (i.e., greater than 30 seconds) between the PCR product pre-hybridization denaturation step and addition of the product to the typing tray containing hybridization buffer, can cause a dot intensity loss of the B allele at the GC locus. The intensity loss may potentially lead to misinterpretation of the results. After denaturation, signal loss is due to primer annealing and elongation that results in duplex formation which blocks hybridization to the GC B probe. We demonstrate the effects of varied elapsed times following the pre-hybridization denaturation step. Furthermore, this study shows that both "snap-cooling" or the addition of EDTA can effectively be used as safeguards in the event that denatured PCR product is not added to the typing trays in a timely manner as outlined in the protocol.
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