基于聚合酶链反应的cDNA分级文库永生化及筛选

D'Esposito M, Mazzarella R, Pengue G, Jones C, D'Urso M, Schlessinger D

Department of Molecular Microbiology, Washington University Medical School, St Louis, MO 63110.

Nucleic Acids Res. 1994 Nov 11;22(22):4806-9. doi: 10.1093/nar/22.22.4806.

Starting from sequences of at least 60 bp, PCR-based screening has been developed to recover cDNAs from libraries without the necessity for hybridization or extensive DNA extraction steps. The method maintains the indefinite availability of even scarce cDNA libraries and provides an estimate of the relative abundance of the mRNA species. Isolation of a cDNA clone can be done in less than a week. cDNAs were isolated that were cognate for fragments of expressed sequences and for an exon predicted from genomic sequence.

从至少60个碱基对的序列开始，已经开发出基于聚合酶链反应（PCR）的筛选方法，用于从文库中回收互补脱氧核糖核酸（cDNA），而无需杂交或大量的脱氧核糖核酸提取步骤。该方法能确保即使是稀缺的cDNA文库也能长期可用，并能估计信使核糖核酸（mRNA）种类的相对丰度。分离一个cDNA克隆可以在不到一周的时间内完成。已分离出与表达序列片段以及从基因组序列预测的一个外显子同源的cDNA。