Computational protocol: Decreased Rate of Evolution in Y Chromosome STR Loci of Increased Size of the Repeat Unit

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[…] Seventeen of the markers analysed (1 tri-, 14 tetra-, 1 penta-, and 1 hexanucleotide STRs) belong to the AmpFlSTR® Yfiler™ Kit; the additional six penta- and one hexanucleotide STRs are reported in , five of them being previously described and two novel.The samples were analysed with the Applied Biosystems AmpFlSTR® Yfiler™ Kit according to the recommendations of the manufacturer on the ABI PRISM® 3130xl Genetic Analyzer (Applied Biosystems, California, USA). The results were analysed using the ABI PRISM® program GeneMapper® 4.0 (Applied Biosystems).The rest of the markers analysed in this study were found screening the human Y chromosome sequence in the GenBank database for penta- and hexanucleotide repeats, using Alex Dong Li's program RepeatFinder 0.4 (unfortunately no longer available, but there are similar programs, such as Tandem Repeats Finder, http://tandem.bu.edu/trf/trf.html) and looking for non-interrupted stretches of eight or more repeats. 41 Y-specific STRs were identified, 19 of them failed to amplify. Of the 22 remaining markers, 5 (Y PENTA 1, DYF411S1, DYS594, DYS596, Y PENTA 2) were analysed in a multiplex system, and 2 more (DYS643, DYS645) were genotyped for this study. The markers DYF411S1, DYS594, DYS596, DYS643, and DYS645 were previously described , whereas Y PENTA 1 and 2 were novel. The repeat units of the 7 penta- and hexanucleotide markers, the primers used to amplify them, and the GenBank accession numbers for the amplified regions are reported in . The forward primers of the five markers analysed in the multiplex system were labelled with fluorescent dyes at the 5′ ends: Y PENTA 1 and DYF411S1 with 6-FAM, DYS594 and DYS596 with HEX, and Y PENTA 2 with TAMRA.The five STR markers amplified with fluorescence-labelled forward primers (Y PENTA 1, DYF411S1, DYS594, DYS596, Y PENTA 2) were amplified in a multiplex system under the following conditions: 1.25 µl GeneAmp PCR Buffer II without MgCl2, 1.5 µl MgCl2 (25 mM), 0.25 µl dNTP mix (10 mM), 2 µl PCR primer mastermix (individual primer concentrations 0.07–1.5 µM), 0.1 µl AmpliTaq Gold (5 U/µl), 6.4 µl ddH2O and 1 µl template DNA (1–10 ng/µl) were mixed per sample (total reaction volume 12.5 µl), and PCR cycling was performed as follows: 95°C, 10 min; 30 cycles (94°C, 30 sec; 60°C, 1 min; 72°C, 1 min); 65°C, 45 min; end at 10°C. Then, 0.5 µl of each PCR product and 0.15 µl of internal size standard (MegaBACE ET400-R Size Standard) were diluted in 9.5 µl Hi-Di Formamide and loaded directly onto the MicroAmp™ Optical 96-Well Reaction Plate. The samples were run on the ABI PRISM® 3130xl Genetic Analyzer (Applied Biosystems) using the Applied Biosystems Multi-Capillary DS-30 (Dye Set D) Matrix Std Kit as recommended by the manufacturer. The genotyping results were analysed using the ABI PRISM® programs GeneScan® 3.7 and Genotyper® 3.7 (both from Applied Biosystems).Two STR markers (DYS643, DYS645) were amplified without fluorescent labels in separate PCR reactions under the following conditions: 1.5 µl GeneAmp PCR Buffer II without MgCl2, 1.2 µl MgCl2 (25 mM), 0.15 µl dNTP mix (10 mM), 2×0.3 µl PCR primer solution (10 µM each), 0.15 µl FIREPol® DNA Polymerase I (5 U/µl), 10.4 µl ddH2O and 1 µl template DNA (1–10 ng/µl) were mixed per sample (total reaction volume 15 µl) and PCR cycling was performed as follows: 94°C, 3 min; 40 cycles (94°C, 25 sec; 55°C, 30 sec; 72°C, 35 sec); 72°C, 3 min; end at 4°C. The products were sequenced using the Applied Biosystems BigDye® Terminator v3.1 Cycle Sequencing Kit as recommended by the manufacturer on the ABI PRISM® 3730xl DNA Analyzer (Applied Biosystems). The sequencing results were analysed using the program ChromasPro. […]

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