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Robino C.,University of Turin | Ralf A.,Erasmus University Rotterdam | Pasino S.,University of Turin | De Marchi M.R.,University of Turin | And 24 more authors.
Forensic Science International: Genetics | Year: 2015

Recently introduced rapidly mutating Y-chromosomal short tandem repeat (RM Y-STR) loci, displaying a multiple-fold higher mutation rate relative to any other Y-STRs, including those conventionally used in forensic casework, have been demonstrated to improve the resolution of male lineage differentiation and to allow male relative separation usually impossible with standard Y-STRs. However, large and geographically-detailed frequency haplotype databases are required to estimate the statistical weight of RM Y-STR haplotype matches if observed in forensic casework. With this in mind, the Italian Working Group (GEFI) of the International Society for Forensic Genetics launched a collaborative exercise aimed at generating an Italian quality controlled forensic RM Y-STR haplotype database. Overall 1509 male individuals from 13 regional populations covering northern, central and southern areas of the Italian peninsula plus Sicily were collected, including both "rural" and "urban" samples classified according to population density in the sampling area. A subset of individuals was additionally genotyped for Y-STR loci included in the Yfiler and PowerPlex Y23 (PPY23) systems (75% and 62%, respectively), allowing the comparison of RM and conventional Y-STRs. Considering the whole set of 13 RM Y-STRs, 1501 unique haplotypes were observed among the 1509 sampled Italian men with a haplotype diversity of 0.999996, largely superior to Yfiler and PPY23 with 0.999914 and 0.999950, respectively. AMOVA indicated that 99.996% of the haplotype variation was within populations, confirming that genetic-geographic structure is almost undetected by RM Y-STRs. Haplotype sharing among regional Italian populations was not observed at all with the complete set of 13 RM Y-STRs. Haplotype sharing within Italian populations was very rare (0.27% non-unique haplotypes), and lower in urban (0.22%) than rural (0.29%) areas. Additionally, 422 father-son pairs were investigated, and 20.1% of them could be discriminated by the whole set of 13 RM Y-STRs, which was very close to the theoretically expected estimate of 19.5% given the mutation rates of the markers used. Results obtained from a high-coverage Italian haplotype dataset confirm on the regional scale the exceptional ability of RM Y-STRs to resolve male lineages previously observed globally, and attest the unsurpassed value of RM Y-STRs for male-relative differentiation purposes. © 2014 Elsevier Ireland Ltd. Source

Fattorini P.,University of Trieste | Previdere C.,University of Pavia | Sorcaburu-Cigliero S.,University of Trieste | Marrubini G.,University of Pavia | And 33 more authors.
Electrophoresis | Year: 2014

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 μg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification "relative" to the used kit (probe) is possible, being the "absolute" amount of DNA inversely related to the length of the target region (r2 = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped-out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Barbaro A.,Studio Indagini Mediche e Forensi SIMEF | Cormaci P.,Studio Indagini Mediche e Forensi SIMEF
Forensic Science International: Genetics Supplement Series | Year: 2015

Y-chromosome specific STRs are commonly analyzed by forensic laboratories for paternity, genealogical testing and forensic caseworks, in particular in sexual assault cases where the identification of male specific DNA is essential in presence of mixed samples containing high female DNA quantity.PowerPlex® Y23 System [1] is a 5-dye multiplex kit that analyze the 17 Y-STR loci commonly available in other kits (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4) together with 6 new discriminating Y-STR loci (DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) that have higher gene diversities.Promega amplification protocol recommends a PCR total reaction volume of 25. μL containing 5.0. μL Master Mix, 2.5. μL Primer Mix, 7.5. μL Grade Water and 10. μL template DNA.The purpose of the present study is to validate the PowerPlex® Y23 System using half volume reaction mix for the amplification of a wide range of forensic samples.Results demonstrate the PowerPlex® Y23 System is a robust and sensitive kit capable of overcoming common inhibitors, giving reliable amplification results also with male/female DNA mixtures and low amounts of DNA template. © 2015 Elsevier Ireland Ltd. Source

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