DIRECT SEQUENCING OF TERMINAL REGIONS OF GENOMIC P1 CLONES - A GENERAL STRATEGY FOR THE DESIGN OF SEQUENCE-TAGGED SITE MARKERS

A method for the preparation of P1 DNA is presented, which allows the direct sequencing of ends of inserts in genomic P1 clones using the Ap plied Biosystems 373A DNA Sequencer and the Dye Terminator sequencing methodology. We surveyed several common methods of DNA preparation inc luding alkaline lysis, Triton-lysozyme lysis, CsCl density-gradient pu rification, and a commercial column matrix DNA purification kit manufa ctured by Qiagen. We found that a modified alkaline lysis preparation of P1 DNA was most successful for generating P1 DNA that could be sequ enced directly. We also noted that the host bacterial strain from whic h the P1 DNA was purified dramatically affected the quality of sequenc ing templates. The bacterial strains NS3145 and NS3529, in which the D rosophila melanogaster and human P1 genomic libraries are harbored, ro utinely yielded poor-quality sequencing templates. However, the bacter ial strain DH10B routinely yielded P1 DNA that was sequenced successfu lly. A bacterial mating scheme is presented that exploits ya transposi tion events to allow the transfer of P1 clones from the library host s train to DH10B. Using either an SP6 or a T7 primer, an average of 350 base pairs of DNA sequence was obtained with an uncalled base frequenc y of similar to 2%. About 4% of P1 end sequences generated corresponde d to unique Drosophila loci present in the Genbank database. These sin gle-pass DNA sequences were used to design sequence-tagged site marker s for physical mapping studies in both humans and Drosophila.