Generation of cohesive ends on PCR products by UDG-mediated excision of dU, and application for cloning into restriction digest-linearized 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 SacI or PstI ends is demonstrated by the subcloning of a 383 to 388-bp fragment of bovine basic fibroblast growth factor into restriction enzyme-linearized pT7T318U. UDG-mediated cohesive ends imperfectly matched to PstI-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 min at 37 degrees C) 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 5' overhang cohesive ends on PCR fragments.
Smith
C
C
University Department of Clinical Biochemistry, Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, UK.
Day
P J
PJ
Walker
M R
MR
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics; D045647:DNA Glycosylases; D003857:Deoxyuridine; D016222:Fibroblast Growth Factor 2 / Q000235:genetics*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D051981:Uracil-DNA Glycosidase
D016222:Fibroblast Growth Factor 2; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D003857:Deoxyuridine