Isolation of Large-Terminal Sequences of BAC Inserts Based on Double-Restriction-Enzyme Digestion Followed by Anchored PCR

Large insert libraries are critical in genome-related research. Bacterial artificial chromosome (BAC) libraries are widely used in plant, animal, and human research; the ends of BAC clones are used as probes for chromosome walking and to confirm overlapping of contigs, as well as RFLP markers for mapping. Several methods have been developed to isolate BAC ends, including subcloning methods, plasmid rescue, and polymerase chain reaction (PCR)-based methods such as inverse PCR, thermal asymmetric interlaced PCR (TAIL-PCR), and Vectorette PCR (1 , and references therein). Here, we described BAC end isolation based on double-restriction-enzyme digestion followed by anchored PCR. The BAC vector in the examples used here is pBeloBAC11 (2 ). Other vectors can be used, but the specific designation of left or right ends as used here may be different depending on the particular vector used.