Kim H S, Smithies O
Laboratory of Medical Genetics and Genetics, University of Wisconsin, Madison 53706.
Nucleic Acids Res. 1988 Sep 26;16(18):8887-903. doi: 10.1093/nar/16.18.8887.
The modification of chromosomal genes by homologous recombination between exogenous DNA and a target locus provides a powerful approach to the study of gene function. One of the current limitations of this gene targetting is the difficulty of identifying cells containing the correctly modified target locus when the modified gene is not amenable to either direct or indirect selection. We here describe a procedure for identifying correctly modified cells that depends on amplifying by the polymerase chain reaction (PCR) predictable fragments of DNA present only in the desired recombinants. This recombinant fragment assay can detect targetted modification using only a few cells, either alone or mixed with tens of thousands of unmodified cells; it does not depend on the phenotype of the modified gene, or on its expression in the target cells. The PCR amplification needed for the procedure is carried out with a heat stable polymerase and a simple automatic temperature-shift apparatus which is described.
通过外源DNA与靶位点之间的同源重组对染色体基因进行修饰，为基因功能研究提供了一种强大的方法。这种基因靶向目前的局限性之一是，当修饰基因既不适合直接选择也不适合间接选择时，难以鉴定出含有正确修饰靶位点的细胞。我们在此描述一种鉴定正确修饰细胞的方法，该方法依赖于通过聚合酶链反应（PCR）扩增仅存在于所需重组体中的可预测DNA片段。这种重组片段检测仅使用少量细胞（单独或与数万个未修饰细胞混合）就能检测到靶向修饰；它不依赖于修饰基因的表型，也不依赖于其在靶细胞中的表达。该方法所需的PCR扩增使用热稳定聚合酶和所描述的简单自动变温装置进行。
