Prashar Y, Weissman S M
Department of Genetics, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06510, USA.
Proc Natl Acad Sci U S A. 1996 Jan 23;93(2):659-63. doi: 10.1073/pnas.93.2.659.
We have developed an approach to study changes in gene expression by selective PCR amplification and display of 3' end restriction fragments of double-stranded cDNAs. This method produces highly consistent and reproducible patterns, can detect almost all mRNAs in a sample, and can resolve hidden differences such as bands that differ in their sequence but comigrate on a gel. Bands corresponding to known cDNAs move to predictable positions on the gel, making this a powerful approach to correlate gel patterns with cDNA data bases. Applying this method, we have examined differences in gene expression patterns during T-cell activation. Of a total of 700 bands that were evaluated in this study, as many as 3-4% represented mRNAs that are upregulated, while approximately 2% were down-regulated within 4 hr of activation of Jurkat T cells. These and other results suggest that this approach is suitable for the systematic, expeditious, and nearly exhaustive elucidation of subtle changes in the patterns of gene expression in cells with altered physiologic states.
我们开发了一种通过选择性PCR扩增和展示双链cDNA的3'端限制性片段来研究基因表达变化的方法。该方法能产生高度一致且可重复的模式，可检测样本中几乎所有的mRNA，还能分辨隐藏的差异，比如那些序列不同但在凝胶上迁移率相同的条带。对应已知cDNA的条带会在凝胶上移动到可预测的位置，这使得该方法成为一种将凝胶模式与cDNA数据库相关联的强大手段。应用此方法，我们研究了T细胞激活过程中基因表达模式的差异。在本研究中评估的总共700条带中，多达3 - 4%代表上调的mRNA，而在Jurkat T细胞激活后4小时内，约2%的mRNA被下调。这些以及其他结果表明，该方法适用于系统、快速且几乎详尽地阐明生理状态改变的细胞中基因表达模式的细微变化。
