Matsunaga H, Kohara Y, Okano K, Kambara H
Central Research Laboratory, Hitachi Ltd., Tokyo, Japan.
Electrophoresis. 1996 Dec;17(12):1833-40. doi: 10.1002/elps.1150171207.
A new method for selecting and amplifying a single DNA fragment from a mixture is proposed. This method is applicable for the rapid classification of DNA fragments from a mixture and for preparation of sequencing templates. DNAs of several to tens of kilobases (kb) are digested with a four-base recognition restriction enzyme to produce smaller fragments. The complementary strand extension reactions are then carried out to produce fluorophore-labeled DNA fragments from the digestion products. These fragments can be rapidly classified according to their terminal-base sequences and their sizes are analyzed by capillary-array gel electrophoresis (CAGE). Electropherograms are used to characterize the fragments and to select polymerase chain reaction (PCR) primers. Any fragment in a digestion mixture can be amplified by PCR with a pair of primers selected from a primer pool by referring to the electropherograms of the fragments. This method was successfully used to compare the electropherograms of two different DNA strands and to sequence a several-kb DNA fragment without subcloning. Combined with CAGE, this method could be used to dramatically simplify DNA fragment analysis.
提出了一种从混合物中选择并扩增单个DNA片段的新方法。该方法适用于混合物中DNA片段的快速分类以及测序模板的制备。用一种识别四碱基的限制性内切酶消化几千碱基到几十千碱基（kb）的DNA，以产生较小的片段。然后进行互补链延伸反应，从消化产物中产生荧光团标记的DNA片段。这些片段可根据其末端碱基序列快速分类，其大小通过毛细管阵列凝胶电泳（CAGE）进行分析。电泳图用于表征片段并选择聚合酶链反应（PCR）引物。通过参考片段的电泳图，从引物库中选择一对引物，用PCR可扩增消化混合物中的任何片段。该方法成功用于比较两条不同DNA链的电泳图，并对几千碱基的DNA片段进行测序而无需亚克隆。与CAGE相结合，该方法可用于显著简化DNA片段分析。
