Bamhi restriction enzyme

For the genomic DNA isolation, we employed 150 µL of gastric homogenate extracted using the DNeasy Blood and Tissue Kit (Qiagen, Germany) according to the manufacturer’s instructions. We evaluated the CYP2C19*2 (rs4244285; 681G>A and CYP2C19*3 (rs4986893; 636G>A) by polymerase chain reaction (PCR)-restriction fragment length polymorphism. We employed genomic DNA as a template with specific primers and conditions, as previously described [16 (link),34 (link)]. Amplification of CYP2C19*2 resulted in a 168 bp band, and amplification of CYP2C19*3 resulted in a 119 bp band. The amplified PCR products of CYP2C19*2 and *3 were digested with SmaI restriction enzyme (New England Biolabs, Tokyo, Japan) for 1 h at 25 °C and BamHI restriction enzyme (Takara, Tokyo, Japan) for 1 h at 30 °C, respectively. The digested product was checked by agarose gel electrophoresis stained with ethidium bromide. Given that the restriction site is absent in the mutated alleles, the PCR products are not digested by the enzyme.

Nuclear proteins were prepared according to the ChIP protocol. 500 U of the BamHI restriction enzyme (TaKaRa, Japan) was added to the DNA-protein cross-linked sample and digested overnight at 37 °C. The next day, 10% SDS was added to the digested samples, which were incubated at 65 °C for 20 min to inactivate the restriction enzyme. The sample was diluted 1:10 using the DNA Ligation Kit (TaKaRa), incubated at 16 °C for 4.5 h, and further incubated at room temperature for 30 min. Next, 300 mg of proteinase K (Beyotime) and 300 mg of RNase A (Beyotime) was added to the sample and incubated at 65 °C overnight. Then digested DNA was purified and pelleted using the DNA Purify Kit (Millipore) and amplified by qRT-PCR.