Short tandem repeat (STR) loci are small segments of repetitive DNA sequences three to seven base pairs in length which display highly polymorphic regions of the human genome (1,2). The small size of these loci facilitates DNA amplification by the polymerase chain reaction (PCR) (3,4). Development of simultaneous amplification of several such STR loci, known as multiplex PCR (5,6) allows for rapid human identification based on DNA polymorphisms. Detection and analysis of multiplexed PCR products may be conducted on platforms such as capillary (7,8) and flat bed gel electrophoresis (9) with concordant results. Since very small amounts of DNA are required even in a highly degraded form (10,11), this procedure has found many applications in forensic sciences, paternity testing, and other related fields where human identification is necessary (12-14). Lins and colleagues (15) have previously reported the development of the highly discriminating eight-locus PowerPlex™ 1.1 STR multiplex system. In this study we describe an additional ninelocus STR multiplex, PowerPlex™ 2.1, and the pentanucleotiderepeat locus Penta D. The STR loci of these systems can be currently analyzed with three amplification reactions (one for each of the PowerPlex™ 1.1, PowerPlex™ 2.1, and Penta D monoplex, respectively) and quality control is monitored by confirmation of three locus profiles, TPOX, TH01, and vWA, that both multiplex systems include. The PowerPlex™ 2.1 multiplex that is studied here utilizes the same fluorescent detection system as described for PowerPlex™ 1.1 (15) and includes eight tetranucleotide-repeat loci, FGA,

Validation studies that meet TWGDAM (The Working Group on DNA Analysis Methods) and CAC (California Association of Criminalists) guidelines for RFLP (restriction fragment length polymorphism) analysis were performed with the DNA probe EFD52 (D17S26). These studies demonstrate that the probe EFD52 is suitable for forensic casework. No unexpected DNA banding patterns were obtained from controlled studies examining various tissues, sample consistency over many gels, mixtures of body fluids, various substrates, various contaminants and non-human DNA sources. Of all the animal DNAs tested, only one higher primate yielded a single band to EFD52 hybridization. The sensitivity of EFD52 was shown to be comparable to that of other forensic probes. Population frequency distribution tables were prepared from over 4000 alleles and two-locus studies were conducted on nine forensically useful probes. Black, White, Hispanic and Lumbee Indian populations were found to be in Hardy-Weinberg and linkage equilibrium. Comparisons between victim blood standards and epithelial fractions of mixed stains from sexual assault cases were used to demonstrate the robustness of the EFD52 probe in forensic casework.