Full-length single-gene cDNA libraries: applications in splice variant analysis.
Alternative splicing of pre-mRNA may generate many distinct proteins from a single gene: regulation of alternative exon selection constitutes control of molecular structure downstream of transcription. Identifying natural splice variants among hundreds or thousands of theoretical alternatives, and examining the regulation of exon selection at multiple sites, may require screening many full-length cDNAs. We describe methods for preparing full-length cDNA libraries comprising the splice variants from single genes. The methods employ robust long distance reverse transcription, gene-specific second strand synthesis, long PCR, and cloning: with these methods cDNAs coding full-length open reading frames were prepared for 21 ion channels (1.2-15 kb). Exon combinations in isolated clones are determined by multiplex PCR. Approximately 85% of the clones contain full-length inserts. Screening can detect even rare variants (0.1%) in linear proportion to their abundance in initial mRNA pools. Tissue-specific expression patterns are reproducible. We describe methods for quantifying and minimizing artifactual exon recombination by template switching. These methods can be used to generate thousands of full-length clones of even large transcripts (>8 kb) for the systematic identification of splice variants and the analysis of regulation of alternative exon selection.
Regan
M R
MR
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. mregan@welchlink.welch.jhu.edu
Emerick
M C
MC
Agnew
W S
WS
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D015723:Gene Library; D005821:Genetic Techniques; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000235:genetics; D016133:Polymerase Chain Reaction; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D051381:Rats
D017931:DNA Primers; D018076:DNA, Complementary; D007473:Ion Channels; D012322:RNA Precursors