Denaturant gradient gel electrophoresis (DGGE) enables insight into the diversity of the studied microbial communities on the basis of separation of PCR amplification products according to their nucleotide sequence composition. However, the success of the method is accompanied by the inherent appearance of various sequence artifacts that bias the impression of community structure by generating additional bands representing no virtual microbes. PCR-DGGE artifacts require optimization of the method when aiming at the phylogenetic identification of the selected DGGE bands. The aim of our study was to develop a procedure which will increase the reliability of the identification. Samples of rumen fluid were used for the optimization since they contain a complex microbial community that supports the generation of artifactual bands. An optimized procedure following band excision and elution of microbial DNA is proposed including nuclease treatment, selection of DNA polymerase with proofreading activity, and cloning prior to sequencing and identification analysis.

Biotechnical Faculty Animal Science Department Chair for Microbiology and Microbial Biotechnology University of Ljubljana Groblje 3 1230 Domžale Slovenia

Zobrazit více v PubMed

PCR Methods Appl. 1993 Dec;3(3):186-94 PubMed

Appl Environ Microbiol. 1997 Dec;63(12):4645-50 PubMed

Appl Environ Microbiol. 2004 Mar;70(3):1263-70 PubMed

J Microbiol Methods. 2005 Jan;60(1):1-11 PubMed

Appl Environ Microbiol. 2001 Jan;67(1):469-72 PubMed

Nucleic Acids Res. 2002 May 1;30(9):2083-8 PubMed

Folia Microbiol (Praha). 2004;49(2):151-5 PubMed

Microb Ecol. 2007 Jul;54(1):141-50 PubMed

FEMS Microbiol Rev. 1997 Nov;21(3):213-29 PubMed

Cell. 1979 Jan;16(1):191-200 PubMed

Appl Environ Microbiol. 1998 Mar;64(3):907-13 PubMed

FEMS Microbiol Ecol. 2010 Dec;74(3):623-30 PubMed

PCR Methods Appl. 1991 Aug;1(1):17-24 PubMed

Appl Environ Microbiol. 2001 Feb;67(2):880-7 PubMed

FEMS Microbiol Lett. 2004 Mar 19;232(2):153-63 PubMed

Appl Environ Microbiol. 1997 Apr;63(4):1489-97 PubMed

Appl Environ Microbiol. 2004 Apr;70(4):2129-36 PubMed

Eur J Biochem. 2001 Jan;268(1):7-14 PubMed

BMC Microbiol. 2001;1:5 PubMed

Appl Environ Microbiol. 1993 Mar;59(3):695-700 PubMed

Nucleic Acids Res. 1997 Dec 15;25(24):4876-82 PubMed

J Mol Biol. 1990 Oct 5;215(3):403-10 PubMed

Biotechnol Bioeng. 2002 Apr 20;78(2):119-30 PubMed

Microbiol Rev. 1995 Mar;59(1):143-69 PubMed

Appl Environ Microbiol. 1996 Feb;62(2):625-30 PubMed

Annu Rev Microbiol. 2003;57:369-94 PubMed