| 夏乐先 孙文娟 沈振 梁昱婷 柳建设 陈建华 邱冠周.煮沸裂解法和试剂盒法提取浸矿菌基因组DNA的比较[J].现代生物医学进展英文版,2014,14(1):31-35. |
| 煮沸裂解法和试剂盒法提取浸矿菌基因组DNA的比较 |
| Comparison of Genomic DNA Extraction fromBioleaching Acidophilesby Two Methods: Boiling and Kit Methods |
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| DOI： |
| 中文关键词: 煮沸裂解法 试剂盒法 浸矿细菌 聚合酶链反应 实时荧光定量PCR |
| 英文关键词: Boiling methods Kit method Leaching bacteria PCR Real-time PCR |
| 基金项目:国家自然科学基金项目（20803094）；中国博士后特别资助（2012T50710）；湖南省科技计划项目（2011RS4068） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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. |
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