TY  - JOUR
A1  - Iwahana, H
A1  - Tsujisawa, T
A1  - Katashima, R
A1  - Yoshimoto, K
A1  - Itakura, M
T1  - 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.
Y1  - 1994/08/01 
JF  - Genome Research 
JO  - Genome Research 
SP  - 19 
EP  - 25 
VL  - 4 
IS  - 1 
UR  - http://genome.cshlp.org/content/4/1/19.abstract 
N2  - 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. 
ER  -