PNNL

Abstract

The ability to generate partially-digested DNA fragments in a fast fashion is highly desirable for applications such as subcloning, restriction mapping, and detection of polymorphisms. A novel method of producing partial digestion of DNA fragments by PCR with 5-methyl-dCTP is recently reported. PCR products generated in the presence of 5-methyl-dCTP are partially-modified with 5-methyl-dCTP. There are many restriction enzymes that do not cut 5-methyl-dCTP modified restriction enzyme sites. Consequently, when such partially-modified PCR products are digested to completion with 5-methyl-dCTP sensitive restriction enzymes, the result of the restriction digestion will be similar to that of a partial digestion. There is no need to adjust the amount of the enzyme used or the length of the digestion time to obtain partial digestion like conventional method. Here we have tested the use of Pfu DNA polymerase (Promega) for PCR amplification in the presence or absence of 5-methyl-dCTP in the reaction mixture. The PCR products were digested with 5-methyl-dCTP sensitive enzymes such as AluI, CfoI, and HpaII from Promega.