S. Hadi

A novel multiplex assay for simultaneously analysing 13 rapidly mutating Y-STRs

A multiplex polymerase chain reaction (PCR) assay (RM-Yplex) was developed which is capable of simultaneously amplifying 13 recently introduced rapidly mutating Y-STR markers (RM Y-STRs). This multiplex assay is expected to aid human identity testing in forensic and other applications to improve differentiating unrelated males and allow separating related males. The 13 RM Y-STR markers included in the multiplex are: DYF387S1, DYF399S1, DYF403S1ab, DYF404S1, DYS449, DYS518, DYS526ab, DYS547, DYS570, DYS576, DYS612, DYS626 and DYS627. This study reflects the proof of concept to analyse all currently known RM Y-STRs simultaneously and describes the optimization of the multiplex assay. The RM-Yplex assay generated complete RM Y-STR profiles down to 62.5pg of male template DNA, and from male-female DNA mixtures at all ratios tested. We herewith introduce and make available for widespread use in forensic and anthropological studies, an effective and sensitive single multiplex assay for simultaneous genotyping of 13 RM Y-STRs.

Population genetic data on 15 autosomal STRs in a Pakistani population sample.

Allele frequencies for the 15 STR loci (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, VWA, D8S1179, TPOX and FGA) were investigated using PowerPlex((R))16 amplification kit in a sample of 328 unrelated individuals from Islamic Republic of Pakistan. The allelic distributions were in Hardy Weinberg equilibrium for all loci except for the loci FGA, TPX and THO1. Various forensic indices were calculated and a comparison was made with geographically nearby populations.

Development of internal amplification controls for DNA profiling with the AmpFℓSTR(®) SGM Plus(®) kit.

DNA extracted from forensic samples can be degraded and also contain co‐extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpFℓSTR® SGM Plus® kit. The co‐amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC90 and IAC410) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX‐labelled primers. Co‐amplification of IAC90 and IAC410 was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC90 and IAC410 were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC90 signal was still present after all human DNA loci fail to amplify; in contrast, the IAC410 signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles.