一种经济高效的酵母菌落PCR模板DNA制备方法的建立  

Development of a cost-effective method for preparation of template DNA for yeast colony PCR

作　　者：刘微 戴凌燕[2] 苗青 彭辉 张东杰[1] 李志江[1,3] LIU Wei;DAI Ling-yan;MIAO Qing;PENG Hui;ZHANG Dong-jie;LI Zhi-jiang(School of Food Science,Heilongjiang Bayi Agricultural University,Daqing 163319,Heilongjiang Province,China;不详)

机构地区：[1]黑龙江八一农垦大学食品学院,黑龙江大庆163319 [2]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163319 [3]黑龙江省杂粮加工及质量安全工程技术研究中心,黑龙江大庆163319

出　　处：《中国生物制品学杂志》2022年第7期848-852,共5页Chinese Journal of Biologicals

基　　金：黑龙江省自然科学基金联合引导项目(LH2020C086);黑龙江省省属高等学校基本科研业务费科研项目黑龙江八一农垦大学校内基础培育计划(ZRCPY201912);黑龙江八一农垦大学研究生创新科研项目(YJSCX2019-Y54)。

摘　　要：目的建立一种经济高效、结果稳定的酵母菌落PCR模板DNA制备方法。方法将乙酸乙酯处理与高温破壁相结合,建立乙酸乙酯-高温破壁法制备酵母菌落PCR模板DNA,并以此方法对酵母进行目的基因扩增与阳性转化子的筛选。同时,以酵母基因组DNA及高温破壁法制备的模板作为对照。结果乙酸乙酯-高温破壁法制取的模板可直接用于菌落PCR扩增目的基因片段以及筛选阳性转化子,与提取酵母基因组DNA相比,采用乙酸乙酯-高温破壁法制取模板的准确率可达90%以上,而高温破壁法制备模板结果不稳定。结论本实验建立的方法简单高效,结果稳定,成本低廉,可满足大规模筛选阳性转化子。Objective To develop an economical,effective and stable method for preparation of template DNA for yeast colony PCR.Methods The ethyl acetate-high temperature wall breakage method for preparation of template DNA for yeast colony PCR was developed by combining ethyl acetate treatment with high temperature wall breakage,and used for the amplification of target gene and the screening of positive transformants of yeast,using yeast genomic DNA and the template prepared by high temperature wall breakage method as controls.Results The template prepared by ethyl acetate-high temperature wall breakage method could be directly used for colony PCR to amplify the target gene and screen the positive transformants.The accuracy rate of PCR results using the template prepared by the method reached more than 90%as compared with those using the extracted yeast genomic DNA.However,the PCR results using the template prepared by high temperature wall breakage method were unstable.Conclusion The developed ethyl acetatehigh temperature wall breakage method is simple,effective,stable and economic,which meets the requirement for largescale screening of positive transformants.

关 键 词：酵母 菌落PCR 模板DNA 基因组DNA 阳性转化子 

分 类 号：TS201.3[轻工技术与工程—食品科学]