Isolation of large terminal sequences of BAC inserts based on double-restriction-enzyme digestion followed by anchored PCR

Isolation of the terminal portions of genomic DNA cloned in bacterial artif icial chromosomes (BACs) is an important step in map-based cloning, and sev eral methods have been developed. Here, we present a new method based on do uble-restriction-enzyme digestion followed by anchored PCR. BAC DNA was dig ested with two enzymes: NotI and one of four enzymes (EcoRV, HpaI, StuI, or XmnI) that produce blunt termini. After dephosphorylation, these digestion s were ligated to NotI- and EcoRV-digested pMSK, a new cloning vector devel oped in this work that is derived from pBluescript SK(+). PCR products repr esenting the left- and right-terminal sequences of BAC inserts were obtaine d using a primer complementary to pMSK and a primer complementary to sequen ces in either the left arm or the right arm of the BAC vector pBeloBAC11. W e have tested this method with 15 different BAC clones, and PCR products re presenting both the left- and right-terminal sequences have been obtained f rom all 15 BAC clones. This method is simple, fast, reproducible, and uses the same set of primers for any restriction enzyme used. With some modifica tions, it can also be used for isolating the terminal portions of genomic D NA cloned in yeast artificial chromosomes and P1-derived artificial chromos omes.