title: An end-trimming method to amplify adjacent cDNA fragments by PCR.
abstract: 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.
authors.affiliation: Otsuka Department of Clinical and Molecular Nutrition, School of Medicine, University of Tokushima, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Iwahana
authors.forename: N
authors.initials: N
authors.lastname: Mizusawa
authors.forename: S
authors.initials: S
authors.lastname: Ii
authors.forename: K
authors.initials: K
authors.lastname: Yoshimoto
authors.forename: M
authors.initials: M
authors.lastname: Itakura
mesh_terms: D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats
chemical_list: D018076:DNA, Complementary