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

• Published in: BioTechniques Vol. 34; no. 4; pp. 768 - 773
• Main Authors: Hawkins, Phillip R, Jin, Pei, Fu, Glenn K
• Format: Journal Article
• Language: English
• Published: Natick, MA Future Science Ltd 01.04.2003; Eaton; Taylor & Francis Group
• Subjects: Biological and medical sciences; Diverse techniques; DNA - biosynthesis; DNA - genetics; Fundamental and applied biological sciences. Psychology; Molecular and cellular biology; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Messenger - chemistry; RNA, Messenger - genetics; Transcription, Genetic; Messenger RNA; Complementary DNA; Enzyme; Proofreading; Polymerization; Hydrolases; Esterases; Calf thymus ribonuclease H; Method; Secondary structure; Reverse transcription polymerase chain reaction
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/03344st06

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.