Improved Method for the Construction of Cosmid Sublibraries from Yeast Artificial Chromosomes

摘要

We describe here a simple and rapid procedure for the construction of cos-mid sublibraries from human chromosomal fragments cloned in yeast artificial chromosomes (YACs). Subcloning of human DNA fragments in cosmids is increasingly important for the analysis of the human genome. The ultimate goal of many genome projects is the identification of disease-associated genes. Various techniques have been described for the identification of genes from chromosomes or chromosomal regions, including the use ofCG-islands, hybridization selection and exon trapping (2,4,5) The availability of DNA fragments cloned in cosmid vectors is a necessary prerequisite for the efficient application of several gene identification strategies, most notably of exon trapping,which was crucial for the identification of numerous disease genes (e.g., neurofibromatosis I) (7). Since YACs represent frequently the starting material in positional cloning projects, the subcloning into cosmids is frequently the bottleneck of these experiments, Although the subcloning is theoretically an easily performed experiment, in practice, this step is time-consuming and frequently inefficient.