Temasek Life Sciences Laboratory, National University of Singapore, Republic of Singapore.
Yeast. 2011 Mar;28(3):205-12. doi: 10.1002/yea.1831. Epub 2010 Nov 22.
A novel reverse genetic approach termed 'marker reconstitution mutagenesis' was designed to generate mutational allelic series in genes of interest. This approach consists of two simple steps which utilize two selective markers. First, using one selective marker, a partial fragment of another selective marker gene is inserted adjacently to a gene of interest by homologous recombination. Second, random mutations are introduced precisely into the gene of interest, together with the reconstitution of the latter selective marker by homologous recombination. This approach was successfully tested for several genes in the fission yeast Schizosaccharomyces pombe. It circumvents the problems encountered with other methods and should be adaptable to any organism that incorporates exogenous DNA by homologous recombination.
一种新型的反向遗传学方法，称为“标记重建诱变”，旨在产生感兴趣基因中的突变等位基因系列。该方法由两个简单的步骤组成，利用两个选择性标记。首先，使用一个选择性标记，通过同源重组将另一个选择性标记基因的部分片段插入到感兴趣的基因旁边。其次，通过同源重组精确地将随机突变引入感兴趣的基因中，同时重建后者的选择性标记。该方法已在裂殖酵母 Schizosaccharomyces pombe 的几个基因中成功测试。它规避了其他方法中遇到的问题，并且应该适用于通过同源重组整合外源 DNA 的任何生物体。
