A new method to construct a full-length cDNA library of human normal bladder tissue

Objective: Using template-switch mechanism at the 5′-end of mRNA technique (SMART) to construct a full-length cDNA library of human normal bladder tissue. Methods: The novel procedures used the template-switching activity of powerscript reverse transcriptase to synthesize and anchor first-strand cDNA in one step. Following reverse transcription, 5 cycles of PCR were performed using a modified oligo (dT) primer and an anchor primer to enrich the full-length cDNA population with 1.0 g human normal bladder poly(A) ⁺ RNA, then double-strand cDNA was synthesized. After digestion with sfiI and size-fractionation by CHROMA SPIN-400 columns, double-strand cDNA was ligated into λTripIEx₂ vector and was packaged. We determined the titer of the primary library and the percentage of recombinant clones and finally amplified the library. Results: The titer of the cDNA library constructed was 2.1 × 10⁶ pfu · mL^-1, and the amplified cDNA library was 6 × 10¹¹ pfu · mL ^-1, the percentage of recombination clones was 99%. Conclusion: Using SMART technique helps us to construct full-length cDNA library with high efficiency and high capacity which lays solid foundation for screening target genes of bladder diseases with probes and antibodies.