PCR with end trimming and cassette ligation: a rapid method to clone exon-intron boundaries and a 5'-upstream sequence of genomic DNA based on a cDNA sequence.
We described a method for PCR amplification of unknown flanking genomic DNA fragments. This method is a combination of PCR with "end-trimming method" and "cassettes and cassette-primers method". In this method, genomic DNA was digested with three different groups of restriction enzymes. DNA in group 1 was digested with BamHI, BglII, FbaI, or MboI. DNA in group 2 was digested with BlnI, NheI, SpeI, or XbaI. DNA in group 3 was digested with SalI or XhoI. Digested DNA in each group was end-trimmed with Klenow fragment of DNA polymerase I in the presence of only one dNTP; dGTP, dCTP, and dTTP for group 1, 2, and 3, respectively. The synthesized cassettes, C1, C2, and C3, had 5'protruding sequences of 5'-ATC-3',5'-TAG-3', and 5'-CGA-3', respectively. Each compatible cassette was ligated to the end-trimmed DNAs in group 1-3, respectively. Nested PCR was then performed using an end-trimmed and cassette-ligated DNA as a template. Primers annealing to known sequences and cassettes were used for the nested PCR. The amplified DNA fragments were electrophoresed on a polyacrylamide gel and purified. The sequences of the DNA fragments were determined after cloning into pBluescript.
Iwahana
H
H
Otsuka Department of Clinical and Molecular Nutrition, University of Tokushima, Japan.
Tsujisawa
T
T
Katashima
R
R
Yoshimoto
K
K
Itakura
M
M
D000582:Amidophosphoribosyltransferase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes; D018076:DNA, Complementary; D005091:Exons; D016678:Genome; D007438:Introns; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D051381:Rats; D016415:Sequence Alignment
D018076:DNA, Complementary; D000582:Amidophosphoribosyltransferase; D004262:DNA Restriction Enzymes