A Study Of Quadriplex PCR With Chimeric Primers For STR Loci And Validation In Forensic Science

Objective. The short tandem repeat (STR) loci in human genome are important markers for Forensic DNA typing. The purposes of our work are to establish a simplified method of multiplex PCR based on chimeric primers for STR loci, to develop a set of fluorescent quadriplex STR system for forensic DNA typing based on this method, and to validate the forensic application of the system under the guidelines of TMGDAM (The Technology Working Group on DNA Analysis Methods ) in order to address concerns presented in today's legal environment. Methods. A set of chimeric primers and universal primers were designed by us to carry out a multiplex PCR for four STR loci. Using our primer set, four STR loci, D1S1612, D9S2026, D20S161 and D6S477 were amplified and detected by polyacrylamide gel electrophoresis and gels were sliverstained. Also,these STR loci were amplified by multiplex PCR using our primer set, but the universal primers were fluorescent-labeled. The products of the multiplex PCR were genotyped by ABI PRISM 310 Genetic Analyzer. The quadriplex STR systems were evaluated under varies conditions. Validation study of Forensic Science included concordance studies, sensitivity, reproducibility, and species specificity, as well as performance of typing forensic samples. Result. A method of multiplex PCR based on both chimeric and universal primers was established. A fluorescent quadriplex STR system, including D1S1612, D9S2026, D20S161 and D6S477, was developed on the basis of the multiplex PCR. The reaction conditions of multiplex PCR were shown to be optimal yet robust enough to withstand moderate variations. Genotyping with 0.25 ng was successful. Alleles at four STR loci could be detected in mixture sample with a proportion of 1 to 10. Environmental and matrix source analyses revealed an ability of our fluorescent quadriplex STR system to obtain complete genotypes in all samples except those exposed to 80癈 for 12-48 days. Conclusion. The multiplex PCR based on both chimeric and universal primers is an efficient method to amplify STR loci. It is achieved easily and reproducibly by simple adjustment of the individual primer concentrations. The use of chimeric primers provides a method for primer design that eliminates the multiple optimization steps involved in developing multiplex PCR. Our fluorescent quadriplex STR system can be analyzed by capillary electrophoresis and provide a newsources of typing regent for this technique platform with laser detecting system. The validation study showed that our fluorescent quadriplex STR system satisfied the TMGDAM guideline and it can be used for forensic casework.