A new method for specific cleavage of megabase-size chromosomal DNA by lambda-terminase.

Abstract

The development of methods for cleavage of DNA at specific site(s) that are widely spaced would facilitate physical mapping of large genomes. Several methods for rare and specific cleavage of chromosomal DNAs require a nearly complete methylation of a given type of restriction site except the one that is specifically protected. It is expected that as the target DNA increases in length, it will become less likely to achieve nearly complete methylation. The intron-encoded endonucleases may also provide a capability to cleave megabase-sized DNA segments due to their very large recognition sequences. However, there are endogenous cleavage sites in the chromosomes of most organisms. We present here a new method to specifically cleave intact chromosomal DNA using lambda-terminase. A plasmid containing two specific cleavage sites (cohesive-end sites) for lambda-terminase was specifically introduced into the E.coli genome and into chromosome V of S.cerevisiae. Chromosomal DNA was prepared from the resulting strains, and then cleaved with lambda-terminase. The results showed that the 4.7-megabase pair (Mb) circular E.coli chromosome and the 0.58-Mb linear yeast chromosome V were specifically cleaved at the desired sites with very high efficiencies. The approach of using the lambda-terminase cleavage reaction is a simple one-step procedure with a high specificity which is particularly suitable for mapping very large genomes of eucaryotes.