Zhang S S, Chen D, Lu Q
State Key Laboratory of Non-Food Biomass Enzyme Technology, National Engineering Research Center for Non-Food Biorefinery, Guangxi Key Laboratory of Biorefinery, Guangxi Academy of Sciences, Nanning, Guangxi Province, China.
Genet Mol Res. 2012 May 21;11(2):1532-43. doi: 10.4238/2012.May.21.10.
Current protocols to extract genomic DNA from microorganisms are still laborious, tedious and costly, especially for the species with thick cell walls. In order to improve the effectiveness of extracting DNA from microbial samples, a novel protocol, defined as two-step extraction method, along with an improved tissue-grinding device, was developed. The protocol included two steps, disruption of microbial cells or spores by grinding the sample together with silica sand in a new device and extraction of DNA with an effective buffer containing cell lysis chemicals. The device was prepared by using a commercial electric mini-grinder, adapted with a grinding stone, and a sample cup processed by lathing from a polytetrafluoroethylene rod. We tested the method with vegetative cells of four microbial species and two microbial spores that have thick cell walls and are therefore hard to process; these included Escherichia coli JM109, Bacillus subtilis WB600, Sacchromyces cerevisiae INVSc1, Trichoderma viride AS3.3711, and the spores of S. cerevisiae and T. viride, respectively, representing Gram-positive bacteria, Gram-negative bacteria, yeast, filamentous fungi. We found that this new method and device extracted usable quantities of genomic DNA from the samples. The DNA fragments that were extracted exceeded 23 kb. The target sequences up to about 5 kb were successfully and exclusively amplified by PCR using extracted DNA as the template. In addition, the DNA extraction was finalized within 1.5 h. Thus, we conclude that this two-step extraction method is an effective and improved protocol for extraction of genomic DNA from microbial samples.
目前从微生物中提取基因组DNA的方法仍然繁琐、耗时且成本高昂，特别是对于那些具有厚细胞壁的物种。为了提高从微生物样本中提取DNA的效率，我们开发了一种新方法，即两步提取法，并改进了组织研磨装置。该方法包括两个步骤：首先在新装置中将样本与硅砂一起研磨，以破坏微生物细胞或孢子；然后用含有细胞裂解化学物质的有效缓冲液提取DNA。该装置是通过使用商业电动微型研磨机，适配研磨石，并使用聚四氟乙烯棒通过车床加工制成样本杯。我们用四种具有厚细胞壁且因此难以处理的微生物的营养细胞和两种微生物孢子测试了该方法；这些包括大肠杆菌JM109、枯草芽孢杆菌WB600、酿酒酵母INVSc1、绿色木霉AS3.3711，以及分别代表革兰氏阳性菌、革兰氏阴性菌、酵母、丝状真菌的酿酒酵母和绿色木霉的孢子。我们发现这种新方法和装置能够从样本中提取出可用量的基因组DNA。提取的DNA片段超过23 kb。以提取的DNA为模板，通过PCR成功且特异性地扩增出了长达约5 kb的目标序列。此外，DNA提取在1.5小时内完成。因此，我们得出结论，这种两步提取法是一种从微生物样本中提取基因组DNA的有效且改进的方法。
