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

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.).