Generation of vector-insensitive dig-labeled probes from large cosmid library inserts using PCR

We have devised a general method for producing vector-insensitive probes fr ont clones in which insert DNA (ca. 40 kb) could not be amplified in one pi ece nor be excised from the vector sequence. The method involves PCR and ve ctor-specific primers in combination with restriction digestion and ligatio n. It yields specific PCR products that could subsequently be labeled using DIG-11-dUTP in a single-cycle PCR. In colony blot hybridization, the probe s were specific for the insert DNA from which the probe was derived and, im portantly, did not detect vector sequences.