Mészáros M, Morton D B
University of Arizona, Tucson 85721, USA.
Biotechniques. 1996 Mar;20(3):413-9. doi: 10.2144/19962003413.
Subtractive hybridization has been widely used for the identification of differentially expressed genes. Here we describe a simple, sensitive strategy of subtractive hybridization that involves binding the driver poly(A)+ RNA pool to paramagnetic Dynabeads Oligo (dT)25. After hybridization with target cDNA, the molecules common to both pools are removed. The subtracted cDNA is then amplified with PCR and used for library screening. Using this method, we have identified four cDNA clones that represent developmentally regulated transcripts in the central nervous system of the tobacco hornworm Manduca sexta. All four transcripts are of low abundance, comprising only 0.001%-0.5% of the poly(A)+ RNA pool.
消减杂交已被广泛用于鉴定差异表达基因。在此，我们描述了一种简单、灵敏的消减杂交策略，该策略涉及将驱动者聚腺苷酸加尾RNA库与顺磁的磁珠寡聚脱氧胸苷（dT）25结合。与目标cDNA杂交后，去除两个库共有的分子。然后用聚合酶链反应（PCR）扩增消减后的cDNA，并用于文库筛选。利用这种方法，我们鉴定出了四个cDNA克隆，它们代表烟草天蛾Manduca sexta中枢神经系统中受发育调控的转录本。所有这四个转录本丰度都很低，仅占聚腺苷酸加尾RNA库的0.001% - 0.5%。
