Full-length cDNA synthesis for long-distance RT-PCR of large mRNA transcripts.
Reverse transcription of mRNA often leads to the synthesis of partial, non-full-length 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 misincorporation 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.
Hawkins
Phillip R
PR
Incyte Genomics, 3160 Porter Drive, Palo Alto, CA 94304, USA.
Jin
Pei
P
Fu
Glenn K
GK
D004247:DNA / Q000096:biosynthesis* / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic
D012333:RNA, Messenger; D004247:DNA