Efficient Second-Strand cDNA Synthesis Using T7 DNA Polymerase

摘要

ABSTRACTWe present a second-strand cDNA synthesis method that takes advantage of both the very high processivity and the very high 3′ exonuclease activity of T7 DNA polymerase. The first strand is synthesized with reverse transcriptase using oligo(dT) as a primer. After alkaline hydrolysis of the mRNA template, a tract of dT residues is synthesized with terminal transferase at the 3′ end of the first strand. The second strand is synthesized using oligo(dA) as a primer. Several oligo(dA) molecules probably anneal to the poly(dT) tract. Because the 3′ exonuclease activity of T7 DNA polymerase is very high, the region of the tract annealed to these oligo(dA) molecules is digested. However, the region of the tract annealed to the very oligo(dA) molecule used as a primer for second-strand synthesis is protected. The resulting cDNA molecules could be cloned with a high efficiency. The size distribution of cloned c-myccDNAs was estimated by Southern blot analysis of phage DNA prepared from the amplified library and by analysis of isolated clones. The results indicate that this method allows to obtain full-length cDNA clones with a high effic