Fargnoli J, Holbrook N J, Fornace A J
Laboratory of Molecular Genetics, NIA, NIH, Baltimore, Maryland 21224.
Anal Biochem. 1990 Jun;187(2):364-73. doi: 10.1016/0003-2697(90)90471-k.
A hybridization subtraction protocol that uses low ratios of RNA to cDNA has been developed to enrich for the cDNA of transcripts that are elevated in one cell population relative to another. This low-ratio hybridization subtraction protocol was found to yield substantial enrichment for the cDNA of low-abundance transcripts induced or increased only several fold. Conditions for the cloning of cDNA enriched by our hybridization subtraction and identification of clones coding for induced transcripts are presented. By screening the cDNA library with probes synthesized from the starting cDNA and cDNA enriched by low-ratio hybridization subtraction, clones coding for induced transcripts could be efficiently identified. The choice of reverse transcriptase used to synthesize the cDNA was found to be important for the enrichment of cDNA for longer length RNA. Low-ratio hybridization subtraction of cDNA synthesized with MMLV reverse transcriptase was effective for the enrichment of cDNA coding for RNA to at least 5 kb in length, while the AMV enzyme was effective only for the cDNA of shorter RNA (less than 1 kb). The characterization of several different low-ratio hybridization subtraction libraries is presented, and the advantages and disadvantages of various hybridization subtraction strategies are discussed.
一种使用低RNA与cDNA比例的杂交扣除方案已被开发出来，用于富集在一个细胞群体中相对于另一个细胞群体表达上调的转录本的cDNA。发现这种低比例杂交扣除方案能显著富集仅被诱导或增加几倍的低丰度转录本的cDNA。本文介绍了通过我们的杂交扣除富集cDNA并鉴定编码诱导转录本的克隆的条件。通过用从起始cDNA和经低比例杂交扣除富集的cDNA合成的探针筛选cDNA文库，可以有效地鉴定编码诱导转录本的克隆。发现用于合成cDNA的逆转录酶的选择对于富集较长长度RNA的cDNA很重要。用MMLV逆转录酶合成的cDNA进行低比例杂交扣除对于富集编码长度至少为5 kb的RNA的cDNA是有效的，而AMV酶仅对较短RNA（小于1 kb）的cDNA有效。本文介绍了几种不同的低比例杂交扣除文库的特征，并讨论了各种杂交扣除策略的优缺点。
