Preparing Nested-Deletion Template DNA for Field Inversion Gel Electrophoresis Analyses and Position-Specific End Sequencing With Transposon Primers

Several protocols for direct sequencing of bacterial artificial chromosome (BAC) and P1 ends have been described recently (1 ,2 ). Primers located within a BAC or P1 vector have been used to sequence either end of a genomic insert to facilitate selection of minimally overlapping clones for analyzing large chromosomal regions (1 ). The entire insert in a BAC or P1-derived artificial chromosome (PAC) clone, however, is usually sequenced using shotgun procedures coupled with gap filling (3 ). Substantial redundant sequencing of 2-kb subclones appears unavoidable in such approaches. In certain circumstances, however, the sequence of only a small portion of a large BAC or PAC clone is required. Situations such as these arise either when known genetic markers need to be mapped or when new ones need to be identified in a small region of a large clone. Nested deletions become particularly useful in these applications.