Extraction and purification of microbial DNA from soil and sediment samples

被引:180
作者
Roose-Amsaleg, CL [1 ]
Garnier-Sillam, E [1 ]
Harry, M [1 ]
机构
[1] Univ Paris 12, UFR Sci, Lab Biol Sols & Eaux, F-94010 Creteil, France
关键词
soil microorganisms; DNA extraction; DNA purification;
D O I
10.1016/S0929-1393(01)00149-4
中图分类号
S15 [土壤学];
学科分类号
0903 ; 090301 ;
摘要
Knowledge of the microbial diversity in natural ecosystems has long been limited because only a minority of naturally occurring microbes can be cultured using standard techniques. Several protocols for the extraction of nucleic acids directly from the environmental matrix have been recently developed to circumvent this problem and this review covers the major extraction procedures currently used to obtain microbial DNA from environmental samples. DNA extraction procedures can involve cell extraction or direct lysis, depending on whether or not the microbial cells are isolated from their matrix. An extraction protocol generally comprises three steps: cell lysis that can be chemical, mechanical and enzymatic, removal of cell fragments and nucleic acid precipitation and purification. Direct lysis methods are more often used than cell extraction ones because they are less time consuming and give a better recovery, resulting in an extracted DNA more representative of the whole microbial community present in the sample. However, with direct lysis, contaminants are also extracted which interfere with the DNA extract. As a consequence, a more extensive purification step is required. At least four types of purification are commonly used: cesium chloride density gradient ultracentrifugation, chromatography, electrophoresis and dialysis and filtration. To remove all contaminants, it could be recommended that several purification procedures be combined, depending on the environmental matrix. The efficiency of extraction/purification depends on the properties of the environmental sample, and each step of the extraction procedure must be adjusted for each sample. Moreover, each step of the procedure suffers from shortcomings, and each additional step inevitably induces a DNA loss. Thus, the choice of a protocol must be a compromise between the recovery of DNA that will be the most representative of the microbial community and the quality of the DNA obtained that is imposed by the objectives of the work, such as detection of specific organisms or assessment of the total microbial community structure. Nevertheless, molecular techniques, that could be used in combination with cultivation techniques, are powerful methods for surveying the microbial diversity in environmental samples, although investigators must be aware that such techniques are not exempt of methodological biases. (C) 2001 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:47 / 60
页数:14
相关论文
共 92 条
[1]   PHYLOGENETIC IDENTIFICATION AND IN-SITU DETECTION OF INDIVIDUAL MICROBIAL-CELLS WITHOUT CULTIVATION [J].
AMANN, RI ;
LUDWIG, W ;
SCHLEIFER, KH .
MICROBIOLOGICAL REVIEWS, 1995, 59 (01) :143-169
[2]  
Atlas R. M., 1993, MICROBIAL ECOLOGY FU
[3]  
Bakken L. R., 1995, Nucleic acids in the environment., P9
[4]   SEPARATION AND PURIFICATION OF BACTERIA FROM SOIL [J].
BAKKEN, LR .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1985, 49 (06) :1482-1487
[5]   Bacterial diversity in a deep-subsurface clay environment [J].
BoivinJahns, V ;
Ruimy, R ;
Bianchi, A ;
Daumas, S ;
Christen, R .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1996, 62 (09) :3405-3412
[6]   Molecular microbial diversity of an agricultural soil in Wisconsin [J].
Borneman, J ;
Skroch, PW ;
OSullivan, KM ;
Palus, JA ;
Rumjanek, NG ;
Jansen, JL ;
Nienhuis, J ;
Triplett, EW .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1996, 62 (06) :1935-1943
[7]   Molecular microbial diversity in soils from eastern Amazonia: Evidence for unusual microorganisms and microbial population shifts associated with deforestation [J].
Borneman, J ;
Triplett, EW .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1997, 63 (07) :2647-2653
[8]   Rapid and sensitive method for the detection of Mycobacterium chlorophenolicum PCP-1 in soil based on 16S rRNA gene-targeted PCR [J].
Briglia, M ;
Eggen, RIL ;
deVos, WM ;
vanElsas, JD .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1996, 62 (04) :1478-1480
[9]   AMPLIFICATION OF DNA FROM NATIVE POPULATIONS OF SOIL BACTERIA BY USING THE POLYMERASE CHAIN-REACTION [J].
BRUCE, KD ;
HIORNS, WD ;
HOBMAN, JL ;
OSBORN, AM ;
STRIKE, P ;
RITCHIE, DA .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1992, 58 (10) :3413-3416
[10]   Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone libraries [J].
Chandler, DP ;
Fredrickson, JK ;
Brockman, FJ .
MOLECULAR ECOLOGY, 1997, 6 (05) :475-482