Developmental validation of the PowerPlex 16 HS System: an improved 16-locus fluorescent STR multiplex.

STR multiplexes remain the cornerstone of genotyping forensic samples. The PowerPlex 16 HS System contains the core CODIS loci: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, and vWA. Additional loci amplified in the multiplex reaction are the sex-determinant locus, amelogenin, and two pentanucleotide STR loci, Penta D and Penta E. The PowerPlex 16 HS System is an updated version of the PowerPlex 16 System; while the primers and dyes remain unchanged, it introduces an enhanced buffer system that includes hot-start Taq DNA polymerase and ensures robust performance. Due to the modification of the reaction mix, a multi-laboratory developmental validation study was completed to document performance capabilities and limitations for the revised assay. Data within this validation was generated by eight laboratories and served as the basis for the following conclusions: genotyping of single-source samples was consistent across a large range of template DNA concentrations with most laboratories obtaining complete profiles at 62.5pg. Mixture analyses showed that over 90% of minor alleles were detected at 1:9 ratios. Optimum amplification cycle number was ultimately dependent on the sensitivity of the detection instrument and could be adjusted to accommodate a range of DNA template concentrations. Reaction conditions including volume and annealing temperature as well as the concentrations of primers, Taq DNA polymerase, and magnesium were shown to be optimal and able to withstand moderate variations without affecting multiplexed STR amplification. Finally, data from non-probative samples and concordance studies showed consistent results when comparing the PowerPlex 16 HS System with the PowerPlex 16 System as well as other commercially available systems.

Y-STR profiling in extended interval (> or = 3 days) postcoital cervicovaginal samples.

Depending upon specific situations, some victims of sexual assault provide vaginal samples more than 36-48 h after the incident. We have tested the ability of commercial and in-house Y-STR systems to provide DNA profiles from extended interval (> or =3 days) postcoital samples. The commercial Y-STR systems tested included the AmpFlSTR Yfiler (Applied Biosystems), PowerPlex Y (Promega) and Y-PLEX 12 (Reliagene) products whereas the in-house systems comprised Multiplex I (MPI) and Multiplex B (MPB). Three donor couples were recruited for the study. Postcoital cervicovaginal swabs (x2) were recovered by each of the three females at specified intervals after sexual intercourse (3-7 days). Each time point sample was collected after a separate act of sexual intercourse and was preceded by a 7-day abstention period. As a negative control, a precoital swab was also recovered prior to coitus for each sampling and only data from postcoital samples that demonstrated a lack of male DNA in the associated precoital sample was used. A number of DNA profile enhancement strategies were employed including sampling by cervical brushing, nondifferential DNA extraction methodology, and post-PCR purification. Full Y-STR profiles from cervicovaginal samples recovered 3-4 days after intercourse were routinely obtained. Profiles were also obtainable 5-6 days postcoitus although by this stage partial profiles rather than full profiles were a more likely outcome. The DNA profiles from the sperm fraction of a differential lysis were superior to that obtained when a nondifferential method was employed in that the allelic signal intensities were generally higher and more balanced and exhibited less baseline noise. The incorporation of a simple post-PCR purification process significantly increased the ability to obtain Y-STR profiles, particularly from 5- to 6-day postcoital samples. Remarkably an 8 locus Y-STR profile was obtained from a 7-day postcoital sample, which is approaching the reported time limit for sperm detection in the cervix.

Performance characteristics of commercial Y-STR multiplex systems.

In this work, a number of performance checks were carried out to evaluate the efficacy of commercial Y-short tandem repeats (Y-STR) kits for casework applications. The study evaluated the sensitivity, specificity and stability of the Y-STR markers used and the ability to obtain a male profile from postcoital samples taken at various time points after intercourse. All systems performed well with 1-3 ng of male DNA as recommended by the manufacturers. All systems gave full profiles at 100 pg of input DNA, which is within the realm of low copy number DNA analysis. Moreover all, except Y-Plex12, gave full profiles with 30-50 pg of male DNA. No increased performance was obtained with any of the systems by increasing the cycle number beyond that recommended by the various manufacturers. When up to 1 microg of female DNA was used (in the absence of male DNA) no female DNA cross reactivity was observed with the Y-Plex 12 and Y-Filer systems. PowerPlex Y produced female DNA derived products near the DYS438 and within the DYS392 loci at a rare allele position with high input DNA levels (300 ng and 1 microg, respectively). Male/female DNA admixture experiments indicated the particularly high specificity of the Y-Filer and PowerPlex Y systems under conditions of several thousand fold female DNA excess. All systems were able to detect the minor alleles in male/male DNA admixtures at a 1:5 dilution with the PowerPlex Y and Y-Filer being able to detect some minor alleles at 1:20. Species testing indicated some limited, minor cross reactivity of the commercial systems with some domestic male mammals although it is easily recognizable and would not pose any problems in casework analysis. As expected a significant number of cross-reacting products were obtained with nonhuman primate species. All Y-STR multiplex systems tested were able to produce complete Y-STR profiles from bloodstains and semen stains exposed up to 6 weeks when the samples were protected against precipitation and sunlight. However, exposure of the samples to precipitation either in the presence or absence of sunlight resulted in Y-STR profile loss over time, with total profile loss occurring with all systems after 3 weeks or more. Complete Y-STR profiles of the male donors up to 72 h postcoitus were obtained with all of the multiplex systems tested, except for Y-Plex12, which gave partial profiles.