Effects of DNA extraction and universal primers on 16S rRNA gene-based DGGE analysis of a bacterial community from fish farming water

被引：7

作者：

Luo P. ^{[1
]}

Hu C. ^{[1
]}

Zhang L. ^{[1
]}

Ren C. ^{[1
]}

Shen Q. ^{[1
]}

机构：

[1] The Key Laboratory of Applied Marine Biology of Guangdong Province, Chinese Academy of Sciences, South China Sea Institute of Oceanology, Chinese Academy of Sciences

来源：

Chinese Journal of Oceanology and Limnology | 2007年 / 25卷 / 3期

关键词：

bacterial community; DGGE; DNA extraction; universal primers;

D O I：

10.1007/s00343-007-0310-7

中图分类号：

学科分类号：

摘要：

Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE. © 2007 Science Press.

引用

页码：310 / 316

页数：6

共 21 条

[1]

Araya R., Tani K., Takagi T., Yamaguchi N., Nasu M., Bacterial activity and community composition in stream water and biofilm from an urban river determined by fluorescent in situ hybridization and DGGE analysis, FEMS Microbiol. Ecol., 43, pp. 111-119, (2003)

[2]

Dahllof I., Baillie H., Kjekkeberg S., rpoB-based microbial community analysis avoids limitations inherent in 16S rRNA gene intraspecies heterogeneity, Appl. Environ. Microbiol., 66, pp. 3376-3380, (2000)

[3]

Evans F.F., Rosado A.S., Sebastian G.V., Casella R., Machado P., Holmstrom C., Jelleberg S., van Elsas J.D., Seldin L., Impact of oil contamination and biostimulation on the diversity of indigenous bacterial communities in soil microcosms, FEMS Microbiol. Ecol., 48, pp. 1-11, (2004)

[4]

Kalia A., Rattan A., Chopra P., A method for extraction of high-quality and high-quantity genomic DNA generally applicable to pathogenic bacteria, Anal. Biochem., 275, pp. 1-5, (1999)

[5]

Li M., Gong J.H., Cottrill M., Yu H., Lange C., Burton J., Topp E., Evaluation of QIAamp® DNA stool mini kit for ecological studies of gut microbiota, J. Microbiol. Methods, 54, pp. 13-20, (2003)

[6]

Liesack W., Weyland H., Stackebrandt E., Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed-culture of strict basophilic bacteria, Microbiol. Ecol., 21, pp. 191-198, (1991)

[7]

Lipthay J.R., Enzinger C., Johnsen K., Aamand J., Sorensen S.J., Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis, Soil Biol. Biochem., 36, pp. 607-614, (2004)

[8]

Marchesi J.R., Sato T., Weightman A.J., Martin T.A., Fry J., Hiom S.J., Wade W.G., Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA, Appl. Environ. Microbiol., 64, pp. 795-799, (1998)

[9]

Martin-Laurent F., Philippot L., Hallet S., Chaussed R., Germon J.C., Soulas G., DNA extraction from soils: old bias from new microbial diversity analysis methods, Appl. Environ. Microbiol., 67, pp. 354-359, (2001)

[10]

Massana R.M., Pedros-Alio C., Spatial differences in bacterioplankton composition along the Catalan cost (NW Mediterranean) assessed by molecular fingerprinting, FEMS Microbiol. Ecol., 33, pp. 51-59, (2000)

← 1 2 3 →