Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries
Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries
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DOI：
10.1073/pnas.93.12.6025
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发表时间：
1996-06-11
影响因子：
11.1
通讯作者：
Siebert, PD
中科院分区：
文献类型：
--
作者：
Diatchenko, L;Lau, YFC;Siebert, PD
抑制消减杂交（suppression subtractive hybridization，SSH）是近年来发展起来的一种高效的消减cDNA文库制备方法。标准化步骤使靶群体内cDNA的丰度相等，并且减法步骤排除靶群体和驱动群体之间的共同序列。在模型系统中，SSH技术在一轮扣除杂交中富集了超过1，000倍的稀有序列。我们通过构建睾丸特异性cDNA文库和使用消减的cDNA混合物作为杂交探针鉴定人Y染色体粘粒文库中的同源序列来证明其有用性。进一步证实分离的粘粒中的人DNA插入片段以睾丸特异性方式表达。这些结果表明，SSH技术适用于许多分子遗传学和定位克隆研究，用于鉴定疾病，发育，组织特异性，或其他差异表达的基因。
A mew and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries, It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library, The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. Those results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.