Wach A
Institut fur Angewandte Mikrobiologie, Biozentrum, Universitat Basel, Switzerland.
Yeast. 1996 Mar 15;12(3):259-65. doi: 10.1002/(SICI)1097-0061(19960315)12:3%3C259::AID-YEA901%3E3.0.CO;2-C.
A PCR-method for fast production of disruption cassettes is introduced, that allows the addition of long flanking homology regions of several hundred base pairs (LFH-PCR) to a marker module. Such a disruption cassette was made by linking two PCR fragments produced from genomic DNA to kanMX6, a modification of dominant resistance marker making S. cerevisiae resistant to geneticin (G418). In a first step, two several hundred base pairs long DNA fragments from the 5'- and 3'- region of a S. cerevisiae gene were amplified in such a way that 26 base pairs extensions homologous to the kanMX6 marker were added to one of their end. In a second step, one strand of each of these molecules then served as a long primer in a PCR using kanMX6 as template. When such a LFH-PCR-generated disruption cassette was used instead of a PCR-made disruption cassette flanked by short homology regions, transformation efficiencies were increased by at least a factor of thirty. This modification will therefore also help to apply PCR-mediated gene manipulations to strains with decreased transformability and/or unpredictable sequence deviations.
介绍了一种用于快速生产破坏盒的PCR方法，该方法允许在标记模块上添加数百个碱基对的长侧翼同源区域（LFH-PCR）。通过将从基因组DNA产生的两个PCR片段连接到kanMX6（一种使酿酒酵母对遗传霉素（G418）具有抗性的显性抗性标记的变体）来制备这样的破坏盒。第一步，以这样的方式扩增来自酿酒酵母基因5'和3'区域的两个数百个碱基对长的DNA片段：在其一端添加与kanMX6标记同源的26个碱基对的延伸。第二步，然后这些分子中的每一个的一条链在以kanMX6为模板的PCR中用作长引物。当使用这种LFH-PCR产生的破坏盒而不是侧翼有短同源区域的PCR制备的破坏盒时，转化效率提高了至少30倍。因此，这种修饰也将有助于将PCR介导的基因操作应用于转化能力降低和/或存在不可预测序列偏差的菌株。
