Reverse Transcriptase Template Switching: A SMART™ Approach for Full-Length cDNA Library Construction

Abstract

Here, we describe a fast, simple method for constructing fulllength cDNA libraries using SMART™ 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 using a modified oligo(dT) primer and an anchor primer to enrich the cDNA population for fulllength sequences. Starting with 1 μg human skeletal muscle poly(A)+ RNA, a cDNA library was constructed that contained 3 × 106 independent clones with 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® 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.