GasCanBase
A Comprehensive Database for Gastric Cancer-associated Polymorphisms

Blood sampling and DNA extraction

Peripheral blood was drawn in EDTA containing vacutainer (BD, Oxford, UK) Tube. Erythrocytes of the samples were lysed by osmotic shock using 20 mM Tris-HCl (pH 8.0). DNA was extracted using Genomic DNA isolation kit (FavorPrep, Favorgen, Taiwan). The DNA concentration was determined using a Nanodrop spectrophotometer (Thermo Scientific, USA). Acceptable DNA samples (A260/A280 of 1.8-2.0) were diluted to 50 ng/μl and stored at -80°C until used.

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Genotyping

Genotype of the polymorphism (CDH1) was determined by allele specific polymerase chain reaction method using the following primers 5'GCCTTATGATTCTCTGCTCG-3' (Wild Type forward), 5'GCCTTATGATTCTCTGCTCA-3' (Mutant Type forward)  and 5'-AACCACCAGCAACGTGATTT-3' (reverse). Briefly, the PCR mixture contained 1x PCR master mix (Thermo Fisher Scientific, Waltham, MA USA ), 10 picomoles of each primer, 50-100 ng of genomic DNA in a total volume of 25 μl. Amplification was accomplished by following thermal cycling condition: 95°C for 5 minutes (one cycle); 95°C for 30 Seconds, 57°C for 20 Seconds and 72°C for 30 Seconds (30 cycles); 72°C for 7 minutes (one cycle), and hold at 4°C. The amplification product (241 bp) was visualized and documented using Gel documentation (protiensimple, Santa Cara, CA USA) after resolving the product along with DNA size markers (1 Kb+ DNA ladder, Invitrogen, USA) in 2% agarose gel and staining the gel with SYBR Safe DNA gel stain (Thermo Fisher Scientific, USA). 

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Sequencing

The two of the samples which showed heterozygous genotype were subjected to sequencing using Sanger method (ABI 3500).