Applied biosystems 3500 genetic analyzer

Thirty‐eight Y‐STR loci plus 3 Y‐InDels were co‐amplified using the Yfiler™ Platinum PCR Amplification Kit in a GeneAmp PCR 9700 thermal cycler (Thermo Fisher Scientific) following the manufacturer's instructions. Amplified fragments were detected by capillary electrophoresis on the Applied Biosystems 3500 Genetic Analyzer (Thermo Fisher Scientific). The electrophoresis data were automatically analyzed by GeneMapper^® ID‐X software (Thermo Fisher Scientific). Negative control (H₂O) and positive control (007) were genotyped in each batch of DNA amplification. We strictly followed the recommendations for the DNA commission of the International Society of Forensic Genetics (ISFG) in the present study (Gusmao et al., 2006).

Fragment analysis of 27 Y-STR loci (DYS576, DYS389I, DYS635, DYS389II, DYS627, DYS460, DYS458, DYS19, YGATAH4, DYS448, DYS391, DYS456, DYS390, DYS438, DYS392, D YS518, DYS570, DYS437, DYS385, DYS449, DYS393, DYS439, DYS481, DYF387S1, DYS533) was performed using the Yfler Plus Amplification Kit (ThermoFisher Scientific, USA) on a SimpliAmp Thermal Cycler (ThermoFisher Scientific, USA). PCR products were separated by electrophoresis using LIZ600 size standard v2 (ThermoFisher Scientific, USA) in a Hi-Di Formamide Master Mix (ThermoFisher Scientific, USA) on an 8 capillary Applied Biosystems 3500 genetic analyzer (ThermoFisher Scientific, USA). ThermoFisher Scientific’s GeneMapper IDx v.1.6 software was used to examine the electropherograms. Samples with non-standard patterns, off-ladder and microvariant alleles were repeated. Haplotypes were used to determine haplogroups with the Nevgen Y-DNA haplogroup prediction tool [47 ]. Subsequently, genotyping was performed according to 23 Y-SNPs candidate for haplogroups (M174, M35, F1756, M48, F1918, M407, M285, P287, M69, M253, M438, M267, M172, M178, P43, P31, M122, M346, M198, M478, M269, M124, M70) on a QuantStudio5 instrument (ThermoFisher Scientific, USA) using TaqMan assays (ThermoFisher Scientific, USA).

To examine the inhibitory effects of selected adhesive removers on short tandem repeat (STR) profiling we used the Applied Biosystems™ NGM Detect™ PCR Amplification Kit (Thermo Fisher Scientific) [34 ]. The amplification performance of the “Integrated Quality Control System” (IQC) of the kit was used to evaluate the success of the PCR reaction, provide an indication of sample quality and distinguish samples that are degraded from those that contain PCR inhibitors. Male human genomic DNA (2800M Control DNA, Promega, 0.5 ng each) was subjected to PCR in a total reaction volume of 25 μL using the Veriti thermal cycler applying amplification conditions recommended by the manufacturer. For inhibition screening, chemical solvents (S2, S6 and S9, respectively) were substituted for the water component of the PCR mix (0, 0.5, 1.25, 2.5, 5 and 7.5 μL, respectively) to achieve total concentrations of 0, 2, 5, 10, 20 and 30% (v/v), respectively. PCR products were analysed on the Applied Biosystems™ 3500 Genetic Analyzer (Thermo Fisher Scientific) using GeneMapper ID-X software v. 1.6.

DNA was extracted from plant tissue using CTAB method [48 (link)].
MYBL2-1 gene was amplified using primers 5′-AGAGGGAGAAAGTACGCAAAG-3′ and 5′-AGAAGTGTTTCTTGACTCGTTGA-3′. PCR products were purified with ExoSAP-IT™ PCR Product Cleanup Reagent (Thermo Fisher Scientific, Waltham, MA, USA), prepared using BigDye™ Terminator v3.1 Cycle Sequencing Kit and subjected to Sanger sequencing via Applied Biosystems 3500 genetic analyzer (Thermo Fisher Scientific, Waltham, MA, USA).

A total of 25 forensically related genetic makers (CSF1PO, FGA, TH01, TPOX, VWA, D1S1656, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D22S1045, D6S1043, Penta D, Penta E, Amelogenin and one Y-InDel of rs2032678) were coamplified using the Huaxia Platinum PCR amplification system. Multiplex amplification was conducted on a ProFlex^TM PCR system (Thermo Fisher Scientific, USA) following the manufacturer’s protocol. The 25 μL PCR volume contained 10 μL of master mix, 10 μL of primer set, 4 μL of deionized water and 1 μL of template DNA. Thermal cycler conditions were as described below: predenaturation at 95 °C for 1 min, followed by 26 cycles of 94 °C for 3 s, 59 °C for 16 s, and 65 °C for 29 s, and a final extension at 60 °C for 5 min. Separation and analysis of PCR amplified products was performed on the Applied Biosystems 3500 Genetic Analyzer (Thermo Fisher Scientific, USA) using POP-4 polymer (Life Technologies, USA), and injections were conducted at 1.2 kV for 16 s. Allele identification was conducted using the Huaxia Platinum panels, bin sets, stutter files and a 175 relative fluorescence units (RFU) threshold, unless otherwise stated, and were compared with the allele ladder provided by the corresponding kit via Applied Biosystems GeneMapper ID-X version 1.2 software.