PARTIAL CVIJI DIGESTION AS AN ALTERNATIVE APPROACH TO GENERATE COSMIDSUBLIBRARIES FOR LARGE-SCALE SEQUENCING PROJECTS

We demonstrate an alternative method for the generation of random subc lones for large-scale shotgun human DNA sequencing projects. Miniprep DNA from a human cosmid clone was partially digested with CviJI, size- fractionated by agarose gel electrophoresis and cloned into bacterioph age M13. A library consisting of 10(5) subclones of 1.1 kb average siz e was recovered from one gel fraction containing approximately 300 ng of partially digested DNA. DNA sequences from an initial 103 subclones demonstrate that 100 of the subclones cover 90% of the cosmid, which is close to the 92% expected if the sublcones were generated at random . DNA sequences from three of the subclones did not match the cosmid s equences, establishing that miniprep DNA can be used for library const ruction with little concern for contamination with genomic E. coli DNA . The use of CviJI to generate random DNA fragments thus offers a simp le alternative to other commonly used fragmentation methods, such as s hearing or sonication, for the generation of random sublibraries for l arge-scale human shotgun DNA sequencing projects.