An end-trimming method to amplify adjacent cDNA fragments by PCR.

We describe a method for cDNA cloning by PCR, which we named "an end-trimming method." This method can be used for PCR amplification and cloning of unknown cDNA fragments adjacent to a short stretch of a known sequence by using a combination of a sequence-matched primer with an ATCG sequence added to the 5' end (5'-ATCG-primer) and an adaptor-(dT)17-primer (dT-primer). The fragments amplified by PCR using a 5'-ATCG-primer, which were modified to have a 5'-ATC overhang by blocking the G site, were exclusively cloned into pUC19 with the vector having a 3'-TAG-5' complementary overhang with a confined direction of inserted fragments. Practical application of this method for the determination of rat amidophosphoribosyltransferase cDNA resulted in successful cloning of adjacent cDNA fragments.