Development and validation of the AmpFlSTR Yfiler PCR amplification kit: a male specific, single amplification 17 Y-STR multiplex system.

In the past 5 years, there has been a substantial increase in the use of Y-short tandem repeat loci (Y-STRs) in forensic laboratories, especially in cases where typing autosomal STRs has met with limited success. The AmpFlSTR Yfiler PCR amplification kit simultaneously amplifies 17 Y-STR loci including the loci in the "European minimal haplotype" (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393), the Scientific Working Group on DNA Analysis Methods (SWGDAM) recommended Y-STR loci (DYS438 and DYS439), and the highly polymorphic loci DYS437, DYS448, DYS456, DYS458, Y GATA H4, and DYS635 (formerly known as Y GATA C4). The Yfiler kit was validated according to the FBI/National Standards and SWGDAM guidelines. Our results showed that full profiles are attainable with low levels of male DNA (below 125 pg) and that under optimized conditions, no detectable cross-reactive products were obtained on human female DNA, bacteria, and commonly encountered animal species. Additionally, we demonstrated the ability to detect male specific profiles in admixed male and female blood samples at a ratio of 1:1000.

Analysis of non-suspect samples lacking visually identifiable sperm using a Y-STR 10-plex.

Y-STRs are valuable in the investigation of sexual assaults in which autosomal STR genotype interpretation is challenging. To detect male DNA from compromised sexual assault evidence, 45 non-suspect samples were differentially extracted and analyzed with 10 Y-STRs. These samples were positive for the presence of human seminal fluid, but were negative for spermatozoa by microscopic examination. Y-STR data were obtained in approximately 86.2% of the epithelial or sperm fractions. On samples yielding incomplete profiles, results were obtained on an average of 5 loci per sample. The inability to obtain results may be due to insufficient amplifiable male DNA, PCR inhibition, or unfounded accusations of sexual assault. This study indicates that it is possible to obtain a male STR profile even in the absence of visually identifiable spermatozoa. Furthermore, Y-STR loci should become components of CODIS if they are to be used in solving non-suspect sexual assaults.

Validation and uses of a Y-chromosome STR 10-plex for forensic and paternity laboratories.

Y-chromosome short tandem repeats (Y-STRs) provide valuable information in cases of rape and questioned paternity, and they allow for the genetic identification of male lineages. The present study validated a Y-STR 10-plex on the ABI PRISM 3100 Genetic Analyzer for use in forensic and paternity laboratories at Orchid Cellmark. Following optimization of the polymerase chain reaction, father-son pairs were analyzed to ensure that each pair generated identical haplotypes. This study demonstrated that the 10-plex is sensitive to 0.125 ng of input DNA and that female samples mixed with male samples did not interfere with Y-STR haplotyping. In a sample of 525 males, there were three instances of locus multiplication, two at DYS19 and one at DYS435. Overall, haplotype diversity was 0.996, suggesting that the 10-plex can effectively distinguish among male lineages.

Regulation of p53 stabilization by DNA damage and protein kinase C.

We have demonstrated previously that the protein kinase C (PKC) signal transduction pathway acts upstream of caspases to regulate caspase activation and apoptosis induced by the DNA-damaging agent cisplatin (CP). In the present study, we have examined whether PKC influences p53 and, hence, cellular sensitivity/resistance to CP. The basal p53 level was low in HeLa cells but was elevated in CP-resistant HeLa (HeLa/CP) cells. CP had no effect on the p53 content in HeLa cells, but it caused p53 accumulation in HeLa/CP cells. Rottlerin, a PKCdelta inhibitor that prevents CP-induced proteolytic activation of PKCdelta, caused an accumulation of p53 in HeLa cells when treated in conjunction with CP, but it had no additional effect in HeLa/CP cells. The ability of rottlerin to prevent proteolytic activation of PKCdelta or to induce accumulation of p53 by CP was compromised in HeLa/CP cells. PKC activator phorbol 12, 13-dibutyrate attenuated constitutive p53 levels in both HeLa and HeLa/CP cells. Whereas the combination of rottlerin and CP increased the half-life of p53 in HeLa cells, CP alone was sufficient to stabilize p53 in HeLa/CP cells. These results suggest that both DNA damage and inhibition of proteolytic activation of PKCdelta by CP were necessary for the stabilization of p53 in HeLa cells. Furthermore, an increase in p53 was not associated with enhanced sensitivity of HeLa cells to CP.