A novel method in cleavage of chromosomal DNA at a single site has been developed. Saccharomyces cerevisiae genome was precisely cleaved at a predetermined site using Cre/loxP recombination which mediates the sitespecific recombination between two loxP sites composed of 34bp each. At first, the loxP site was introduced into one of the 16 yeast chromosomes. The chromosomes were isolated from the resulting strain in a intact form and reacted with synthetic oligonucleotides containing loxP sites in the presence of Cre recombinase. By the recombination between two loxP sites on a chromosome and a oligonucleotide, the chromosome containing loxP sites was cleaved specifically. The cleavage was confirmed by Southern hybridization. When biotin-labeled oligonucleotides were used in reaction with plasmid DNA, the simultaneous cutting and labeling of DNA fragment could be accomplished.
염색체 DNA를 한 위치에서 절단하고 표지하는 새로운 방법을 개발하였다. Cre단백질은 34bp로 이루어진 loxP 부위에서 site-specific recombination을 일으키는데, 이러한 Cre/loxP recombination을 이용하여 Saccharomyces cerevisiae genome의 특정 위치에서 절단이 일어나도록 하였다. loxP부위를 16개의 효모 염색체 중의 하나에 삽입시킨 다음, 염색체를 완전한 형태로 분리해내어 Cre단백질의 존재하에 loxP 부위를 포함하는 oligonucleotide와 반응시켰다. 또한, biotin으로 표지된 oligonucleotide와 plasmid DNA를 Cre에 의하여 반응시켰을 때, plasmid DNA가 절단과 동시에 표지되는 것을 확인하였다.
