Rapid Amplification of Flanking Sequences of a Known DNA Region by Partial Restriction Digestion and Hot Start PCR  

作　　者：LOU Qun-feng LIU Qiang YANG Yin-gui CHEN Jin-feng 

机构地区：[1]State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing210095, P.R.China

出　　处：《Agricultural Sciences in China》2008年第5期542-546,共5页中国农业科学（英文版）

基　　金：partially supported by the Program 30470120,30671419,30700541 from the National Natural Science Foundation of China;by the 863 Programs 2006AA10Z108,2006AA100108 from the Ministry of Science and Technology of China;by the Ph.D Funding 20050307009 from the Ministry of Education of China;by the Program BK2006139 from the Natural Science Foundation of Jiangsu Province;by Research Fund KJ05006 from Nanjing Agricultural University,China.

摘　　要：A simple and efficient method for cloning the flanking genomic sequences of a known DNA region is reported in this study. This method combined partial restriction endonuclease digestion, adaptor ligation, and a single round polymerase chain reaction. Total genomic DNA was partially digested with the frequent-cutting restriction enzyme Mse I. The partially digested products were ligated to an unphosphorylated adaptor. A hot start PCR amplification with Taq polymerase and dNTP was performed with a DNA-specific primer and the adaptor primer complementary to the adaptor and the Mse I recognition site. The amplified products were fractionated, cloned and sequenced. By this method, we cloned the downstream region of a gynoecious marker TG/CAC234 from cucumber （Cucumis sativus L.）.A simple and efficient method for cloning the flanking genomic sequences of a known DNA region is reported in this study. This method combined partial restriction endonuclease digestion, adaptor ligation, and a single round polymerase chain reaction. Total genomic DNA was partially digested with the frequent-cutting restriction enzyme Mse I. The partially digested products were ligated to an unphosphorylated adaptor. A hot start PCR amplification with Taq polymerase and dNTP was performed with a DNA-specific primer and the adaptor primer complementary to the adaptor and the Mse I recognition site. The amplified products were fractionated, cloned and sequenced. By this method, we cloned the downstream region of a gynoecious marker TG/CAC234 from cucumber （Cucumis sativus L.）.

关 键 词：Cucumis sativus L. Mse I partial digestion sequence cloning hot start PCR 

分 类 号：S642.2[农业科学—蔬菜学]