BioTechniques (Apr 2003)

Full-Length cDNA Synthesis for Long-Distance RT-PCR of Large mRNA Transcripts

  • Phillip R. Hawkins,
  • Pei Jin,
  • Glenn K. Fu

DOI
https://doi.org/10.2144/03344st06
Journal volume & issue
Vol. 34, no. 4
pp. 768 – 773

Abstract

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Reverse transcription of mRNA often leads to the synthesis of partial, non-fulllength cDNAs. Methods to facilitate reverse transcription across RNA regions of secondary structure, as well as enzyme modifications to eliminate RNase H activities inherent to reverse transcriptase enzymes, have been previously reported. However, because all reverse transcriptases have high error rates of polymerization, the mis-incorporation of nucleotides can also cause the reverse transcriptase to stumble. Hence, even in the absence of RNA secondary structure and RNase H activity, the synthesis of full-length cDNA from long mRNA transcripts still remains a challenge. We describe here the coupling of a 3′→5′ exonuclease function during reverse transcription. The incorporation of a proofreading activity, when used in conjunction with denaturant buffers and RNase H-deficient reverse transcriptases, can successfully generate full-length cDNAs of up to 15 kb.