Developmental Validation of Reduced-Size STR Miniplex Primer Sets

The short tandem repeat (STR) multiplex primer sets--Miniplex 2, Miniplex 4, and Big Mini--contain 12 of the 13 CODIS loci, and the primer binding regions of each locus are designed to produce amplicons that are as short as possible, thus providing an effective tool for the analysis of degraded DNA samples. This developmental validation study shows the robustness, reliability, and reproducibility of Miniplex sets 2, 4, and Big Mini under a variety of conditions. DNA template concentrations as low as 100 pg/25ml can be successfully amplified with high sensitivity and good peak balance ratio using 33 cycles. Sensitivity studies, peak balance measurements, and allele concordance data conclusively demonstrated that Miniplex sets produced consistent and reliable results at this level. These studies provide important data for forensic laboratories in assessing the stability of the Miniplex sets under a variety of conditions that may arise in the interpretation of data from complex forensic samples. Further tests are being conducted in order to examine the application of these multiplexes to nonroutine forensic samples such as hair, fingernail scrapings, and low copy number DNA sources. Since these new sets will be used for the analysis of degraded and low-level DNA, the validation studies were performed using 100-125 pg of DNA, the lowest input level at which peak balance, peak intensity, and allele consistency were stable. A range of tests were performed, including studies of concordance with standard multiplex kits, sensitivity and reproducibility, and PCR amplification conditions. In addition, studies of mixtures, nonhuman and environmentally degraded DNA, and simulated forensic samples were performed. 4 tables, 14 figures, and 20 references