Amplitaq 360 gold dna polymerases

A panel of informative STR markers is located within a 2 MB interval around LZTR1, DVL2, and HBB. Linked markers were selected using the UCSC Genome Browser (http://genome.ucsc.edu/). The residual WGA product of cffDNA was used to confirm the haplotyping results. All forward primers were fluorescently labeled at the 5′ end by either fluorescein (Fam) or hexachlorofluorescein (HEX). The PCR was performed in 20 μl reactions, containing 5 μl of WGA product of cffDNA or 50 ng/μl genomic DNA, 10 µl of AmpliTaq 360 Gold DNA Polymerases (Applied Biosystems), and 0.5 pmol/μl of each primer set. Initial enzyme activation and denaturation were performed at 95°C for 10 min, followed by thermal cycling under the following conditions: denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 70°C for 1 min for 25 cycles of amplification. Fragment analysis was performed using GeneScan software and ABI PRISM 3710 Genetic Analyzer (Applied Biosystems). Finally, to detect cfDNA of maternal origin, AmpFLSTR™ Identifiler™ Plus PCR Amplification kit was used as described by the manufacturer (Applied Biosystems).

Whole-genome amplification (WGA) products of cffDNA were used to detect direct mutations using Sanger sequencing combined with short tandem repeat (STR) identifier analysis for a haplotyping-based approach (Asiri et al., 2020 (link); Umair et al., 2020 (link)). For regions flanking mutations in DVL2, LZTR1, HBB, MYO7A, and RNASEH2B genes, the PCR reactions were performed using cffDNA WGA products in 20 μl reaction mixture consisting of 1× PCR buffer, 5 μl of 1/10 μl of the MDA product or genomic DNA, 0.5 pmol/μl of each primer set, and 10 μl AmpliTaq 360 Gold DNA Polymerases (Applied Biosystems). The initial enzyme activation and denaturation were performed at 95°C for 10 min. Thermal cycling was performed under the following conditions: 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 70°C for 1 min. The PCR products were subjected to cycle sequencing using a BigDye Terminator version 3.1 Ready Reaction Cycle Sequencing Kit with the forward primer. The amplified products were loaded onto an ABI Prism 3710XL Genetic Analyzer with the POP- 7 polymer (Barhoumi et al., 2019 (link); Alhamoudi et al., 2020 (link)).