Rothstein R
Methods Enzymol. 1991;194:281-301. doi: 10.1016/0076-6879(91)94022-5.
The methods described in this chapter permit the manipulation of virtually any cloned yeast chromosomal sequence by virtue of the fact that DNA transformed into yeast integrates into the chromosome by homologous recombination. Furthermore, double-strand breaks in transforming DNA stimulate recombination and can be used to target integration events. This allows simple one-step gene disruption methods using yeast selectable markers. The availability of counterselectable markers makes it possible to replace chromosomal sequences with mutant alleles that cannot be directly selected. Finally, these same methods can be used to rescue chromosomal alleles on plasmids for subsequent molecular analysis.
本章所述方法可对几乎任何克隆的酵母染色体序列进行操作，这是因为导入酵母的DNA通过同源重组整合到染色体中。此外，转化DNA中的双链断裂会刺激重组，可用于靶向整合事件。这使得利用酵母选择标记进行简单的一步基因破坏方法成为可能。可反向选择标记的存在使得用不能直接选择的突变等位基因替换染色体序列成为可能。最后，这些相同的方法可用于拯救质粒上的染色体等位基因，以便后续进行分子分析。
