Genemarker

Ninety-six pairs of SSR primers with three bases and above were designed and synthesized by Tianyi Huiyuan Biotech Company, Wuhan, China (Table S1). The PCR reaction system contained 5.0 μL 2 × Taq PCR Master Mix, 1 μL template DNA (20 ng/μL), 0.5 μL of each primer (10 μL/mol), and DNase-/RNase-free deionized water 3.0 μL. Two-stage amplification programs were used. In the first stage, the pre-denaturation at 95 °C for 5 min caused the annealing temperature to gradually decrease from 62 °C to 52 °C, with a total of 10 cycles. The second stage included 25 amplification cycles, and the annealing temperature was 52 °C. In these two stages, the denaturation and extension steps remained unchanged for 30 s at 95 °C and 72 °C, respectively. After the second stage, the final extension was carried out at 72 °C for 20 min. Ninety-six pairs of primers were selected for PCR amplification and detected by 1% agarose gel electrophoresis. Finally, 13 SSR markers were obtained (Table 2). The PCR products were subjected to SSR analysis on an ABI 3730xl instrument, and then the genotype data were read using GeneMarker (Applied Biosystems).

EMAST status was determined using 5 polymorphic tetranucleotide markers (UT5037, D9S242, D20S85, D8S321, and D20S82) [22 (link)]. Genomic DNA extracted from tumors and counterpart normal tissues were PCR-amplified by specific primers for each tetranucleotide marker using Platinum PCR Supermix (Invitrogen, USA) as per the manufacturer’s protocol. PCR was performed using 6-FAM or HEX labeled primers. Cycling conditions were as follows: 95°C for 15 minutes for the initial heat activation; 40 cycles of 94°C for 1 minute, 55 C°–62C° for 1 minute, and 72°C for 30 seconds; and a final extension at 72°C for 10 minutes. Fluorescently labeled fragments generated by PCR were analyzed on an Applied Biosystems 3730xl DNA Analyzer with the GeneMarker (SoftGenetic LLC, PA). PCR products were used for DNA fragment analysis to identify frameshift mutation at tetranucleotide repeats for each locus. When aberrant peaks +/- multiples of 4 nucleotides were observed in the electrophoretograms from tumor as compared to that of the paired non-tumor, the marker was listed positive for frameshift instability. Tumors showing frameshifts in at least 2 tetranucleotide markers compared to paired normal tissue were categorized as EMAST-positive tumors, whereas all others were categorized as EMAST-negative tumors.