Site-specific cleavage of DNA at 8- and 10-base-pair sequences

A method is described for cutting DNA at specific sites that are 8 and 10 base pairs long. The DNA is first treated with a specific methylase, either the restriction-modification enzyme M. Taq I, which converts the 4-base sequence T-C-G-A to T-C-G-mA, or the similar enzyme M. Cla I, which converts the 6-base sequence A-T-C-G-A-T to A-T-C-G-mA-T. The DNA is then cleaved with Dpn I, a restriction endonuclease that recognizes the sequence G-mA-T-C. Dpn I is unique in that it cuts only DNA that is methylated at adenine in both strands of its recognition sequence. In DNAs that are not otherwise methylated at adenine in both strands of the sequence G-A-T-C, cleavage by Dpn I occurs only at the following sequences: in the case of M. Taq I methylation, 5' T-C-G-mA - T-C-G-mA 3' 3' mA-G-C - T-mA-G-C - T 5'; in the case of M. Cla I methylation, 5' A - T-C-G-mA - T-C-G-mA-T 3' 3' T-mA-G-C - T-mA-G-C - T-A 5'. Specific cutting and cloning at these methylase/Dpn I-generated sites is shown experimentally. Further, we describe how the above technique can be extended to generate Dpn I cleavage sites of up to 12 base pairs. In DNA that contains equal amounts of each base distributed at random, 8- and 10-base-pair recognition sequences occur, on the average, approximately once every 65,000 and 1,000,000 base pairs, respectively. Potential applications, including the development of cloning vectors and a rapid method for chromosome walking, are discussed.