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.

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.