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

Here, we describe a fast, simple method for constructing full-length cDNA l ibraries using SMART(TM) technology. This novel procedure uses the template -switching activity of Moloney murine leukemia virus (MMLV) reverse transcr iptase to synthesize and anchor first-strand cDNA in one step. Following re verse transcription, three cycles of PCR are performed rising a modified ol igo(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 clone s 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 gene s contained intact open reading frames. The average length of complete open reading frames was 2.4 kb. Furthermore, 86% of the full-length clones reta ined longer 5' UTR sequences than the longest 5' end deposited in the GenBa nk(R) database. cDNA libraries generated using this method will be useful f or accelerating the collection of mRNA 5' end sequence information, which i s currently very limited in GenBank.