C-G Linker Adaptor PCR Method for Genome Walking

C-G 링커 어댑터 PCR을 이용한 지놈워킹

Genome walking is a par ticular application for identifying sequences of unknown genomic regions adjacent to a known region. Many genome walking methods based on polymerase chain reaction (PCR) are available. Even if earlier techniques suffer from low reproducibility, inefficiency, and non-specificity, improved strategies have been developed. In this study, we present an alternative strategy: the genomic DNA is digested with restriction enzymes. After cytosine overhangs at 5' ends, the fragments are ligated to linker adaptor s had guanine overhang at 3' ends. Then nested PCR is performed. The improvements in this strategy focus on two points. The first is the C tailing method using Pfu polymerase instead of the A tailing method based on nontemplate-dependent terminal transferase activity of Taq polymerase. Therefore unintended modification of target DNA can be prevented without A tailing error. The second point is the use of C/G-specific ligation had advantage in the ligation efficiency compared with A/T-specific ligation. Therefore, the C-G linker PCR method increases ligation efficiency between digested genomic DNA and adaptor DNA. As a result, the quantity of target DNA to amplify by PCR is enriched. We successfully used G-C linker PCR to retrieve flanking regions bordering the phophinothricin resistance gene in genetically modified tobacco (GMO).