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.
Iwahana
H
H
Otsuka Department of Clinical and Molecular Nutrition, School of Medicine, University of Tokushima, Japan.
Mizusawa
N
N
Ii
S
S
Yoshimoto
K
K
Itakura
M
M
D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats
D018076:DNA, Complementary