C-G Linker Adaptor PCR Method for Genome Walking
2015
Seo, H.S., KTand G Research Institute, Daejeon, Republic of Korea | Lee, Y., KTand G Research Institute, Daejeon, Republic of Korea | Jeon, E., KTand G Research Institute, Daejeon, Republic of Korea | Lee, J., KTand G Research Institute, Daejeon, Republic of Korea
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 over - hangs 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 fir st 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 er ror . 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).
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