Generation of Cohesive Ends on PCR Products by UDG-mediated Excision of dU, and Application I'.or Cloning into Restriction D,gest-ltnear, zed Vectors

We have Investigated the use of dU excision by uracil N-glycosylase (UDG) to create cohesive ends on PCR fragments "mimicking" those generated by restriction enzymes. The feasibility of this approach for directional and nondirectional cloning using cohesive ends mimicking Sacl or Pstl ends is demonstrated by the subcloning of a 383 to 388-bp fragment of bovine basic flbroblast growth factor into restriction enzyme-linearized pTTT318U. UDG-mediated cohesive ends imperfectly matched to Pstl-generated vector ends gave reasonable cloning efficiency and accuracy, suggesting that the approach may be extended to mimicry of other restriction enzymes producing 3' overhangs. The rapid and specific excision of dU by UDG (within 30 rain at 37~ has several potential advantages over the use of restriction site-modified primers, including the avoidance of restriction cleavage at internal sites within the PCR product. Also, following ligation, the approach described may be used to prevent subsequent cleavage of the joined DNA segments by the restriction enzyme, that is, by not recreating the restriction enzyme recognition sequence at the junction, which may find application in gene engineering. By adapting the approach to use dU-containing linkers or "vectorettes," the approach may be used for cloning unknown sequences (e.g., by cDNA or genomic library construction) or for mimicking S' overhang cohesive ends on PCR fragments.