Reverse transcriptase template switching: A SMART (TM) approach forfull-length cDNA library construction

摘要

Here, we describe a fast, simple method for constructing full-length cDNA libraries using SMART(TM) technology. This novel procedure uses the template-switching activity of Moloney murine leukemia virus (MMLV) reverse transcriptase to synthesize and anchor first-strand cDNA in one step. Following reverse transcription, three cycles of PCR are performed rising a modified oligo(dr) primer and an anchor primer to enrich the cDNA population for full-length sequences. Starling with 1 mug human skeletal muscle poly (A)(+) RNA, a cDNA library was constructed that contained 3 x 10(6) independent clones with an an average insert size of 2 kb. Sequence analysis of 172 randomly selected clones showed that 77% of cDNA clones corresponding to known genes contained intact open reading frames. The average length of complete open reading frames was 2.4 kb. Furthermore, 86% of the full-length clones retained longer 5' UTR sequences than the longest 5' end deposited in the GenBank(R) database. cDNA libraries generated using this method will be useful for accelerating the collection of mRNA 5' end sequence information, which is currently very limited in GenBank.