A method for screening arrayed cosmid libraries mega insert yeast artificial chromosomes

摘要

Recent advances in the construction of yeast artificial chromosome (YAC) libraries have produced YAC libraries with an average insert size of about one million base pairs (Mbp). The CEPH Mega YAC library has also been partially ordered and mapped (1).Although these YACs allow the rapid cloning of large genomic regions, their size makes it difficult to analyze these regions in more detail (e.g. to find expressed sequences or to fine map interesting regions). Therefore it is often necessary to converta YAC into a cosmid contig to penorm a more detailed analysis. One strategy to convert a YAC into a cosmid contig is to screen a cosmid library directly with the YAC as a probe (2). However, this approach is fraught with several problems: (i) the hybridization has to be done under suppression conditions, i.e. in the presence of large amounts of unlabelled human DNA, because a YAC contains many repetitive elements; (ii) some method to separate the YAC DNA from the other yeast chromosotnes has to be employed because the YAC constitutes only a small portion of the whole yeast DNA; (iii) the hybridization has to be very efficient, because each cosmid will constitute only a small portion of the probe (～4% in the case of a 1 Mbp YAC).