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高温环境样品总DNA直接和间接提取方法的比较
引用本文:刘宏伟,彭谦,李沁元,肖炜,崔晓龙.高温环境样品总DNA直接和间接提取方法的比较[J].微生物学报,2006,46(6):1018-1022.
作者姓名:刘宏伟  彭谦  李沁元  肖炜  崔晓龙
作者单位:1. 云南大学,云南省微生物研究所,云南省生物资源保护与利用重点实验室,昆明,650091
2. 云南大学生命科学教学实验中心,昆明,650091
3. 昆明理工大学生物与化学工程学院,昆明,650224
基金项目:国家自然科学基金;云南省中青年学术与技术带头人培养基金;教育部留学回国人员科研启动基金;云南省自然科学基金;云南省教育厅资助项目
摘    要:分别采用两种环境总DNA直接提取法和一种间接提取法从6种温泉菌席样品中提取总DNA,以DNA粗产物的纯度、能否用于后续PCR扩增及PCR-DGGE(变性梯度凝胶电泳)所反映的微生物多样性为评价指标对两类方法进行比较和评价。研究发现,虽然间接提取法效率低下,但对于高温极端环境中生物量较小的样品,间接法能得到有研究价值的、纯度较高的环境样品总DNA,而直接法得到的DNA量小且不适于PCR扩增操作。在使用这2类方法都能得到可用于研究操作的DNA的情况下,间接提取法能更好的体现环境样品中微生物的多样性。

关 键 词:菌席  环境DNA  间接DNA提取法  PCR-DGGE
文章编号:0001-6209(2006)06-1018-05
收稿时间:2005-12-21
修稿时间:2005-12-212006-04-25

Comparison of direct and indirect environmental DNA extraction methods for samples from a high-temperature environment
LIU Hong-wei, PENG Qian, LI Qin-yuan, XIAO Wei, CUI Xiao-long.Comparison of direct and indirect environmental DNA extraction methods for samples from a high-temperature environment[J].Acta Microbiologica Sinica,2006,46(6):1018-1022.
Authors:LIU Hong-wei  PENG Qian  LI Qin-yuan  XIAO Wei  CUI Xiao-long
Affiliation:1. Yunnan Institute of Microbiology and Key Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming 650091, China;2 .Experiment Center of Life Sciences, Yunnan University, Kunming 650091, China;3. Faculty of Biologic and Chemical Engineering, Kunming University of Science and Technology, Kunming 650224, China
Abstract:Two different direct DNA extraction methods and one indirect DNA extraction method were applied to recover environmental DNA from six mat samples, which were sampled closely around a high-temperature spring vent in Tengchong Rehai of the western Yunnan province. As the criteria, quantity and purity of the extracted crude DNA, the result of PCR amplification for denaturing gradient gel electrophoresis (DGGE) before and after crude DNA purification, and the Shannon-wiener index of DGGE profiles were used to evaluate the direct and indirect DNA extraction methods. For the samples of less biomass, the indirect method yielded available crude DNA with high purity, and the DNA was amplified without purification by PCR for DGGE. The two direct methods extracted less DNA than indirect method from the samples of less biomass, and the crude DNA extraction could not be amplified by PCR. Despite the lower quantity of DNA yield, the indirect DNA extraction method for the other mat samples presented more diverse bacterial community than that of the direct DNA extraction methods according to the Shannon-wiener index of the PCR-DGGE profiles.
Keywords:Microbial mat  Environmental DNA  Indirect DNA extraction method  PCR-DGGE
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