PCR isolation and cloning of novel splice variant mRNAs from known drug target genes.

Alternative splicing of pre-mRNAs is an important mechanism for the generation of vertebrate protein diversity. Unfortunately, the contribution of alternative splicing to protein diversity is currently not well understood because many full-length mRNA sequences have yet to be identified. In this report, we describe the use of RT-PCR to identify and clone 279 novel alternatively spliced mRNAs from 114 well-known drug target genes. Our findings demonstrate the existence of many novel alternatively spliced mRNA transcripts and suggest that many more genes undergo functionally significant alternative splicing than previously thought.

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.