Direct sequencing of terminal regions of genomic P1 clones: A general strategy for the design of sequence-tagged site markers☆

摘要: A method for the preparation of P1 DNA is presented, which allows direct sequencing ends inserts in genomic clones using Applied Biosystems 373A Sequencer and Dye Terminator methodology. We surveyed several common methods including alkaline lysis, Triton-lysozyme CsCl density-gradient purification, a commercial column matrix purification kit manufactured by Qiagen. found that modified lysis was most successful generating could be sequenced directly. also noted host bacterial strain from purified dramatically affected quality templates. The strains NS3145 NS3529, Drosophila melanogaster human libraries are harbored, routinely yielded poor-quality However, DH10B successfully. mating scheme presented exploits gamma delta transposition events to allow transfer library DH10B. Using either an SP6 or T7 primer, average 350 base pairs sequence obtained with uncalled frequency approximately 2%. About 4% end sequences generated corresponded unique loci present Genbank database. These single-pass were used design sequence-tagged site markers physical mapping studies both humans Drosophila.