Full-Length cDNA Synthesis for Long-Distance RT-PCR of Large mRNA Transcripts. (April 2003)

• Record Type: Journal Article
• Authors: Hawkins, Phillip R.; Jin, Pei; Fu, Glenn K.
• Is Part Of: Biotechniques. Volume 34:Number 4(2003)
• Page Start: 768
• Page End: 773
• Publication Date: 2003-04
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/03344st06
• Languages: English
• ISSNs: 0736-6205

Abstract

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.