Regan M R, Emerick M C, Agnew W S
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Anal Biochem. 2000 Nov 15;286(2):265-76. doi: 10.1006/abio.2000.4819.
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.
前体mRNA的可变剪接可从单个基因产生许多不同的蛋白质：可变外显子选择的调控构成转录下游分子结构的控制。在成百上千种理论上的可变剪接中识别天然剪接变体，并在多个位点研究外显子选择的调控，可能需要筛选许多全长cDNA。我们描述了制备包含单个基因剪接变体的全长cDNA文库的方法。这些方法采用稳健的长距离逆转录、基因特异性第二链合成、长PCR和克隆：利用这些方法为21个离子通道（1.2 - 15 kb）制备了编码全长开放阅读框的cDNA。通过多重PCR确定分离克隆中的外显子组合。大约85%的克隆含有全长插入片段。筛选能够检测到初始mRNA库中丰度与比例呈线性关系的罕见变体（0.1%）。组织特异性表达模式具有可重复性。我们描述了通过模板切换对人为外显子重组进行定量和最小化的方法。这些方法可用于生成数千个甚至大转录本（>8 kb）的全长克隆，用于系统识别剪接变体和分析可变外显子选择的调控。
