Digital mapping of bacterial artificial chromosomes by fluorescence in situ hybridization

The bacterial artificial chromosome (BAC) has become the most popular tool for cloning large DNA fragments. The inserts of most BAC clones average 100 -200 kilobases (kb) and molecular characterization of such large DNA fragme nts is a major challenge. Here we report a simple and expedient technique f or physical mapping of BAC inserts. Individual BAC molecules were immobiliz ed on glass slides coated with Poly-L-lysine. The intact circular BAC molec ules were visualized by fluorescence in situ hybridization using BAC DNA as a probe. The 7.4 kb BAC vector was extended to approximately 2.44 kb per m icrometer. Digitally measured linear distances can be transformed into kilo bases of DNA using the extension of BAC vector as a standard calibration. W e mapped DNA fragments as small as 2 kb directly on circular BAC molecules. A rice BAC clone containing both tandem and dispersed repeats was analyzed using this technique. The distribution and organization of the different r epeats within the BAC insert were efficiently determined. The results showe d that this technique will be especially valuable for characterizing BAC cl ones that contain complex repetitive DNA sequences.