煮沸裂解法和试剂盒法提取浸矿菌基因组DNA的比较

目的:在生物浸出中,微生物群落结构分析有着重要意义,而群落分析的基础是提取纯度高、损失少的基因组DNA。为了解决这一问题,本实验通过比较两种较常用的DNA提取方法,煮沸裂解法和试剂盒法,寻找一种灵敏、快速、经济实用的制备浸矿细菌基因组DNA的方法。方法:分别用煮沸裂解法和试剂盒法提取6种浸矿菌的基因组DNA,从所提取的基因组DNA浓度、纯度、回收率和对PCR扩增反应的影响方面比较了两种方法的提取效果;用两种方法来处理不同浓度梯度的一种菌,通过实时定量PCR来比较两种方法的灵敏性。结果:相同处理量(108个)的革兰氏阳性菌(1株)、革兰氏阴性菌(4株)、古菌(1株)经两种方法提取的基因组DNA差异较大,煮沸裂解法所得的6组基因组DNA更纯,其OD260/OD280的值更接近1.8-2.0(纯DNA的OD260/OD280在1.8-2.0之间),前者所提DNA回收率最大可达后者的16.7倍;煮沸裂解法只需较少菌(102个)便能让实时定量PCR检测到所提DNA模板浓度,比试剂盒法灵敏。结论:两种方法提取的基因组DNA均可用于后续的PCR扩增,此外,前者提取的DNA浓度随细菌浓度增加而呈线性增大,而后者随菌浓度增大,所提DNA量增加有限,因此,在生物浸出中微生物基因组DNA的提取可直接采用简单快速的煮沸提取法,为实验节约成本和时间。

Comparison of Genomic DNA Extraction fromBioleaching Acidophiles by Two Methods: Boiling and Kit Methods

Objective:In bioleaching process, the microbial community structure analysis, based on high purity, less loss of genomic DNA extraction, plays an important role. To solve this problemand find out a more sensitive, rapid, economical and practical way to extract genomic DNA from bioleaching acidophiles, two commonly used DNA extraction methods, boiling and Kit, were compared in the study. Methods:Boiling and Kit methods were used to extract genomic DNA from six different leaching bacteria. The extracting effects of the two methods were compared in terms of the concentration, purity and recovery of the extracted genomic DNA and the influence of extracted genomic DNA on PCR (Polymerase Chain Reaction). The sensitivity of the two methods were compared through Realtime PCR where the same mesophilic acidophile was treated with different decimal amounts of cells.Results:The results showed that the genomic DNAextracted fromthe equal amount of bacteria (108 cells) of grampositive bacteria (1 strain), gramnegative bacteria (4 strains), archaea(1strain)bythetwomethodsareverydifferent.ThesixkindsofgenomicDNAextractedwiththeboilingmethodaremuchpurer thanthose extracted with Kit method, and the values of OD260/OD280 of the former are closer to the value of 1.8 to 2.0 ( the OD260/OD280 value of pure DNA falls between 1.8 to 2.0). Recovery of genomic DNA with the former method can be up to 16.7 times that of the latter. While compared to the kit method, the concentration of extracted DNA template could be detected through Real-time PCR with lower concentration of cells (102 cells) could be detected through Real-time PCR when DNA was extracted by boiling method. Thus, boiling method is higher sensitivity than kit method.Conclusion:The DNA extracted by the two methods could be used for subsequent PCR analysis. Additionally, the DNA concentrations with boiling method increase linearly with the increase of cell concentration, while, the increase of DNA concentration with kit method is limited when cell concentrations increase and arrive a high level. Therefore, the microbial genomic DNA extraction can directly use the boiling method to save cost and time in the bioleaching process.