CONSTRUCTION OF 2 NEAR-KILOBASE RESOLUTION RESTRICTION MAPS OF THE 5'-REGULATORY REGION OF THE HUMAN APOLIPOPROTEIN-B GENE BY QUANTITATIVE DNA FIBER MAPPING (QDFM)

Quantitative DNA fiber mapping (QDFM) is a high-resolution technique f or physical mapping of DNA, The method is based on hybridization of fl uorescently labeled DNA probes to individual DNA molecules stretched o n a chemically modified glass surface. We now demonstrate and validate a rapid QDFM-based approach for the mapping of multiple restriction s ites and precise localization of restriction fragments in large genomi c clones. Restriction fragments of a 70-kb P1 clone (P1-70) containing the 5' region of the human apolipoprotein B gene (APOB) were subclone d and mapped along straightened P1-70 DNA molecules, Multicolor fluore scence in situ hybridization (FISH) and digital image analysis allowed us to rapidly position 29 restriction fragments, ranging in size from 0.5 kb to 8 kb, and to map 43 restriction sites. The restriction map obtained by QDFM was in excellent agreement with information obtained by RecA-assisted restriction endonuclease (RARE) cleavage, long-range PCR, and DNA sequence analyses of the P1-70 clone. These data demonstr ate that QDFM is a rapid, reliable method for detailed restriction sit e-mapping of large DNA clones.