Jones D H, Winistorfer S C
Department of Pediatrics, University of Iowa College of Medicine, Iowa City 52242.
Nucleic Acids Res. 1992 Feb 11;20(3):595-600. doi: 10.1093/nar/20.3.595.
We present a novel method for the PCR amplification of unknown DNA that flanks a known segment directly from human genomic DNA. PCR requires that primer annealing sites be present on each end of the DNA segment that is to be amplified. In this method, known DNA is placed on the uncharacterized side of the sequence of interest via DNA polymerase mediated generation of a PCR template that is shaped like a pan with a handle. Generation of this template permits specific amplification of the unknown sequence. Taq (DNA) polymerase was used to form the original template and to generate the PCR product. 2.2 kb of the beta-globin gene, and 657 bp of the 5' flanking region of the cystic fibrosis transmembrane conductance regulator gene, were amplified directly from human genomic DNA using primers that initially flank only one side of the region amplified. This method will provide a powerful tool for acquiring DNA sequence information.
我们提出了一种全新的方法，可直接从人类基因组DNA中对已知片段侧翼的未知DNA进行PCR扩增。PCR要求待扩增的DNA片段两端存在引物退火位点。在该方法中，通过DNA聚合酶介导生成一个形状像带柄平底锅的PCR模板，将已知DNA置于目标序列未表征的一侧。该模板的生成允许对未知序列进行特异性扩增。使用Taq（DNA）聚合酶形成原始模板并生成PCR产物。使用最初仅位于扩增区域一侧的引物，直接从人类基因组DNA中扩增出了2.2 kb的β-珠蛋白基因以及657 bp的囊性纤维化跨膜传导调节因子基因5'侧翼区域。该方法将为获取DNA序列信息提供一个强大的工具。
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