Berg E S, Olaisen B
Department of Virology, National Institute of Public Health, Oslo, Norway.
Biotechniques. 1994 Nov;17(5):896-901.
We describe a general method for making template DNA for sequencing of PCR products. The procedure may be particularly useful for PCR products where minimal sequence information is known or as an alternative to primer walking when sequencing long PCR products. A cassette containing the hybridization site for the M13 sequencing primer is ligated to a sample PCR product. Using one phosphorylated primer specific for the cassette together with one primer specific for the sample PCR product, subsequent PCR amplifies one hybrid construct directionally. This allows utilization of the universal M13 primer when sequencing of one strand after the removal of the complementary strand using lambda-exonuclease.
我们描述了一种制备用于PCR产物测序的模板DNA的通用方法。该方法对于已知最少序列信息的PCR产物可能特别有用,或者在对长PCR产物进行测序时作为引物步移的替代方法。将含有M13测序引物杂交位点的盒式结构连接到样品PCR产物上。使用一条对该盒式结构特异的磷酸化引物和一条对样品PCR产物特异的引物,随后的PCR定向扩增一个杂交构建体。这使得在使用λ外切核酸酶去除互补链后对一条链进行测序时能够使用通用的M13引物。