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Optimization of the DGGE band identification method
Kušar, Darja (Author), Avguštin, Gorazd (Author)

URLURL - Presentation file, Visit http://www.springerlink.com/content/a48w624573471112/fulltext.pdf This link opens in a new window

Abstract
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 nucelotide 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.

Language:English
Keywords:mikrobiologija, metode, elektroforeza, DGGE
Work type:Not categorized (r6)
Tipology:1.01 - Original Scientific Article
Organization:BF - Biotechnical Faculty
Year:2012
Number of pages:301-306
Numbering:Vol. 57, no. 4
UDC:579
ISSN on article:0015-5632
DOI:10.1007/s12223-012-0130-9 Link is opened in a new window
COBISS.SI-ID:3042696 Link is opened in a new window
Views:929
Downloads:139
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Record is a part of a journal

Title:Folia microbiologica
Shortened title:Folia microbiol.
Publisher:Springer
ISSN:0015-5632
COBISS.SI-ID:25470976 This link opens in a new window

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