NDLI: Comparisons of Different Hypervariable Regions of rrs Genes for Use in Fingerprinting of Microbial Communities by PCR-Denaturing Gradient Gel Electrophoresis

Content Provider	PubMed Central
Author	Yu, Zhongtang Morrison, Mark
Copyright Year	2004
Abstract	Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial diversity and community structure, but no systematic comparison has been made of the DGGE profiles obtained when different hypervariable (V) regions are amplified from the same community DNA samples. We report here a study to make such comparisons and establish a preferred choice of V region(s) to examine by DGGE, when community DNA extracted from samples of digesta is used. When the members of the phylogenetically representative set of 218 rrs genes archived in the RDP II database were compared, the V1 region was found to be the most variable, followed by the V9 and V3 regions. The temperature of the lowest-melting-temperature (Tm(L) ) domain for each V region was also calculated for these rrs genes, and the V1 to V4 region was found to be most heterogeneous with respect to Tm(L) . The average Tm(L) values and their standard deviations for each V region were then used to devise the denaturing gradients suitable for separating 95% of all the sequences, and the PCR-DGGE profiles produced from the same community DNA samples with these conditions were compared. The resulting DGGE profiles were substantially different in terms of the number, resolution, and relative intensity of the amplification products. The DGGE profiles of the V3 region were best, and the V3 to V5 and V6 to V8 regions produced better DGGE profiles than did other multiple V-region amplicons. Introduction of degenerate bases in the primers used to amplify the V1 or V3 region alone did not improve DGGE banding profiles. Our results show that DGGE analysis of gastrointestinal microbiomes is best accomplished by the amplification of either the V3 or V1 region of rrs genes, but if a longer amplification product is desired, then the V3 to V5 or V6 to V8 region should be targeted.
Related Links	http://dx.doi.org/10.1128/aem.70.8.4800-4806.2004
Ending Page	4806
Page Count	7
Starting Page	4800
File Format	PDF
ISSN	00992240
e-ISSN	10985336
Journal	Applied and Environmental Microbiology
Issue Number	8
Volume Number	70
Language	English
Publisher	American Society for Microbiology
Publisher Date	2004-08-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Biotechnology Food Science Ecology Applied Microbiology and Biotechnology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Ecology Food Science Applied Microbiology and Biotechnology Biotechnology

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