PCR-based screening BAC library and direct end sequencing of BAC clones.

A PCR based strategy was developed which required four steps to identify positive BAC clones from barley BAC (bacterial artificial chromosome) library. In the protocol, two levels of BAC DNA pools (super-pool and pool) were prepared for analysis. One pool is made of one plate DNAs and one super-pool is made of mixing ten consecutive 1/100 diluted pool DNAs (1 approximately 10, 11 approximately 20 ect). First,super-pool DNAs were analysed and then 10 pool DNAs contained in every positive super-pool were analysed. Once positive BAC plates were identified,the bacterial cultures were dipped into PCR mixtures and reaction is made to identify positive BAC clones. The BAC clones identified by each marker were grouped into contigs. BAC-end sequence was obtained from BACs within each contig and primers were designed for the next step chromosome walking. In case of the BAC ends belong to repetitive sequence, the primers were designed based on the subcloned unique band in the contig (identified by Hind III digestion pattern). This method allows us to construct the BAC contig without the costly and time-consuming efforts, and no radioactivity harmful to the body.