Zn. Yang et Te. Mirkov, Isolation of large terminal sequences of BAC inserts based on double-restriction-enzyme digestion followed by anchored PCR, GENOME, 43(2), 2000, pp. 412-415
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.