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为同时高效地对大量产黄青霉(Penicillium chrysogenum)突变株进行分子鉴定,本实验探索了一种适合PCR反应的简易产黄青霉基因组提取方法。该方法首先将产黄青霉菌丝转移至0.7 mol/L Na Cl为等渗剂的蜗牛酶(6 mg/m L)和纤维素酶(4 000 U/m L)酶解液中,超声波处理10 min,然后于28℃酶解14 h,最后95℃加热处理10 min。结果表明,应用该方法获得的裂解液上清稀释后可直接作为PCR反应模板,且扩增条带清晰。该方法操作简便,用样量小,可在96孔板一个孔中完成DNA模版的制备过程,并且可同时并行多个样品,适于大规模产黄青霉基因工程突变株的检测,并为其它真菌基因组的提取提供了参考。
Abstract：A simple genomic DNA extraction method was established for simultaneous and high-efficient identification of Penicillium chrysogenum genetic enginnering mutants in macro scale. The P. chrysogenum mycelia were transferred into 0.7 mol/L Na Cl solution containing snailase(6 mg/m L) and cellulose(4 000 U/m L)with the following procedure,ultrasonic treatment 10 min,lyzed at 28℃ for 14 h,heating at 95℃ for 10 min. The PCR results showed the lyzed supenant could be used as the PCR template directly with some dilution. The method is simple and needs small amount sample. The process for template DNA preparation could be completed on 96-well plate and many samples' DNA can be prepared simultaneously. It is suitable for P. chrysogenum genetic engineering mutants identification and also it provides the basis for other fungi's genomic DNA extraction.
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基本信息:
DOI：10.13417/j.gab.034.002255
中图分类号:Q93;Q78
引用信息:
[1]韩梁彦,王丽丽,张春晓.一种适合PCR反应的产黄青霉基因组简易提取方法[J].基因组学与应用生物学,2015,34(10):2255-2261.DOI:10.13417/j.gab.034.002255.
基金信息:
国家科技部863课题——抗生素工业生物过程关键技术集成与示范研究项目(2012AA021204)资助
2015-10-25
2015-10-25