Genescan 120 liz

The MLVA PCR products were diluted 1:80 and subjected to capillary electrophoresis on ABI Prism 3130 Genetic Analyser (Thermo Fisher Scientific, Waltham, USA) with 0.25 µL GeneScan 1200 and sized by GeneMapper 4.0 (Thermo Fisher Scientific, Waltham, USA). Amplified SNR PCR products were diluted 1:80 and subjected to capillary electrophoresis on ABI Prism 3130 Genetic Analyser (Thermo Fisher Scientific, Waltham, USA) with 0.25 µL GeneScan 120 LIZ, and sized by GeneMapper 4.0 (Thermo Fisher Scientific, Waltham, USA). In all the analyses, the samples were processed in triplicate and the concordance of the results allowed the correct sizing of the fragments. Amplified SNR PCR products were diluted 1:80 and subjected to capillary electrophoresis on ABI Prism 3130 Genetic Analyser (Thermo Fisher Scientific, Waltham, USA) with 0.25 µL GeneScan 120 LIZ, and sized by Gene Mapper 4.0 (Thermo Fisher Scientific, Waltham, USA).

Thirty-eight DNA samples provided by CHUSJ were PCR amplified for the polymorphic markers (upstream and downstream of CDH1). Primers used and expected amplicons are described in Supplemental Table S3. For microsatellite analyses, 1 μL PCR products were mixed with 9.5 μL Hi-Di™ Formamide (Applied Biosystems, Foster City, CA, USA) and 0.5 μL of molecular size standard dye GeneScan™ 120 LIZ™ or GeneScan™ 500 ROX™ (Applied Biosystems, Foster City, CA, USA) depending on the molecular size of the amplified fragment. The mixture was run in BI PRISM^® 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and analysed with Peak Scanner™ software (ThermoFisher Waltham, MA, USA). For SNP analysis, the PCR products were purified by FastAP™ thermosensitive alkaline phosphatase and exonuclease I (Thermo Scientific, Wilmington, NC, USA), following the manufacturer’s instructions, and used for Sanger sequencing with the BigDye^® Terminator Cycle v3.1 (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. The purified products were run in ABI PRISM^® 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), and the analysis was performed using Mutation Surveyor^® software (SoftGenetics, State College, PA, USA).

To detect allelic loss at the CDH1 gene (locus 16q22.1) three microsatellite markers were used: D16S3025 at the 5′ end of the CDH1 locus, and D16S496 and D16S3067 at the 3′ end of the CDH1 locus [12 (link)]. Microsatellites were identified in those patients with both tumor and constitutive DNA samples (15 subjects) in a multiplex fluorescent PCR with primers previously described [4 (link)]. Fifty ng of DNA were used as template, and GeneScan™ 120 LIZ™ (Applied Biosystems™) was employed as size standard. The fragments were separated using the ABI PRISM® 310 Genetic Analyzer and the results were analyzed with Peak Scanner™ Software v1.0 (Applied Biosystems™). The LOH was calculated with the following formula: LOH index = (N1/N2)/(T1/T2), corresponding to peak areas of N1 = constitutive DNA allele 1; N2 = constitutive DNA allele 2; T1 = tumor DNA allele 1; and T2 = tumor DNA allele 2. LOH was considered when the LOH index was more than or less than 1.04 ± 0.13 for D16S3025, 1.0 ± 0.67 for D16S496, and 1.06 ± 0.11 for D16S3067 [4 (link)]. LOH-positive results were confirmed by repeated testing.