Page 132 - Helicobacter pylori and Gastric Cancer: From Tumor microenvironment to Immunotherapy

DNA extraction from culture
Genomic DNA of total H. pylori isolates was extracted using the QIAmp DNA mini kit (QIAGEN, Hilden, Germany) according to manufacturer’s protocol and stored at -20 oC until use.
Detection of genes
In this study, PCR was used to detect the H. pylori specific ureC gene for confirming that the cultures represented a bona-fide H. pylori isolate, and the same technique was employed to establish presence or absence of cagE, cagA, iceA1, iceA2 babA2 and babB genes.
All primer sets were selected from published literature (listed in Table 1). PCR reactions were performed in a volume of 50 μL containing 10 mmol/L Tris-HCl, 1.5 mmol/L MgCl2, 0.2 mmol/L of each deoxynucleotide, 25 pmol of each primer and 2.5 units of Taq polymerase (Geneone, Germany)[19, 20]. The thermal cycler program used consisted of the following steps; initial denaturation at 94oC for 3 min followed by 35 cycles of 30 seconds at 94oC (denaturation), 30 seconds at 58oC for cagA and glmM [21]; 30 seconds at 53oC for cagE; 48°C for babA2, 49°C for babB, 57oC For iceA1 and 48oC and for iceA2 all annealing steps, followed by 30 seconds at 72oC (extension step) and a final extension step was 3 min at 72 oC[22].
Chapter 6
Chapter 6
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