Tailored removal of flanking homopolymer sequences from cDNA clones.
Terminal homopolymer sequences introduced during the synthesis and cloning of cDNA molecules often interfere with subsequent expression of the cloned cDNA. We describe a general method for the removal of homopolymer flanking sequences from cDNA inserts and subsequent tailoring of the resulting termini. The cDNA insert containing homopolymer tails is first subcloned into the multiple cloning site of an appropriate transcription vector. cDNA copies are then generated from in vitro-synthesized transcripts using oligonucleotide primers complementary to the nucleotide sequences adjacent to the homopolymer tails. The resulting double-stranded cDNA contains the homopolymer flanking sequences as 3'-terminal extensions that are conveniently removed by the 3'----5' exonuclease activity of T4 DNA polymerase. If the oligonucleotide primers also contain 5'-terminal noncomplementary sequences that specify potential restriction endonuclease sites, those sites are subsequently generated by the 5'----3' polymerase activity of the T4 DNA polymerase. Thus, in the same reaction, flanking homopolymer sequences are removed and the resulting termini are tailored to specify desired sequences.
Xu
Z K
ZK
Department of Cell Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.
Anzola
J V
JV
Nuss
D L
DL
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004274:DNA, Recombinant; D004259:DNA-Directed DNA Polymerase; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012087:Reoviridae / Q000235:genetics*; D013604:T-Phages / Q000201:enzymology
D004274:DNA, Recombinant; D012333:RNA, Messenger; D012367:RNA, Viral; D004247:DNA; D004259:DNA-Directed DNA Polymerase