一种高效快速鉴定酵母转化子的酵母菌落PCR方法

A Rapid and Efficient Yeast Colony PCR Method to Identify Positive Transformants

酵母是一种重要的基因工程宿主菌,但是由于其细胞壁结构复杂牢固,普通的菌落PCR方法的成功率较低。为解决此问题,提供一种快速、简单、高效的酵母菌落PCR方法。该方法先利用溶壁酶溶解酵母细胞壁,然后利用热涨裂解细胞并进行PCR反应。此方法将酵母细胞裂解和PCR在同一个PCR管中完成。试验结果显示此方法能成功扩增目的基因且成功率高。将此方法应用于外源性内切葡聚糖酶(endoglucanase,EG)的酿酒酵母转化子筛选,经与基因组PCR比较,菌落PCR与基因组PCR结果一致。结果证明此方法具有良好的稳定性,适用于酿酒酵母转化子的快速筛选。

Yeast is an important host microorganism in genetic engineering and molecular cloning. However, because of the complex structure of their cell wall, ordinary colony PCR method has a low success rate. To address this problem, a fast, simple and efficient yeast colony PCR method was provided. In the novel method, a commercial available cell wall degradation enzyme, lyticase, was used to disrupt yeast cell wall and the resulted protoblast was then bursted by heating. Into the lysis mixture, other PCR components were added and PCR reactions were performed. In the current constructed method, the yeast cell lysis and PCR reaction were performed in one single PCR tube, which is convenient and easy to implement. The success rate of the novel method was relatively high compared to traditional ones. Using this method, the Saccharomyces cerevisiae transformants of exogenous endoglucanase（EG）were screened. Compared to PCR using genomic DNA, the results of colony PCR were consistent with those from genomic PCR. The results indicated that the novel method was of good stability, repeatability and suitable for the rapid screening of the S. cerevisiae transformants.