Contig assembly of bacterial artificial chromosome clones through multiplexed fluorescence-labeled fingerprinting

Abstract

A rapid multiplexed fingerprinting method has been developed for bacterial artificial chromosome (BAC) contig assembly. Defined subsets of BAC DNA fragments that result from digestion by three paired restriction endonucleases are labeled with unique fluorescent F-ddATP for each subset. Lists of the labeled fragment size are generated by an ABI 377 DNA sequencer and the GeneScan analysis software and then processed by an assembly program, FPC (Fingerprinted Contigs), to produce contig maps. Data obtained from the multiplexed labeling permit detection of smaller overlaps than is observed when data from a single double-digest are analyzed. The method has been tested on 98 BACs from chromosome 22 regions where large-scale sequencing is under way and also through simulation, using randomly generated BAC clones derived from existing DNA sequence data. In each case, contig assembly results demonstrated the advantages of multiplexed fingerprinting.