Denaturing Gradient Gel Electrophoresis Polymorphism for Rapid 16S rDNA Clone Screening and Microbial Diversity Study.

2002 ◽  
Vol 93 (1) ◽  
pp. 101-103 ◽  
Author(s):  
WEN-TSO LIU ◽  
CHUN-LIN HUANG ◽  
JIANG YONG HU ◽  
LIANGFA SONG ◽  
SAY LEONG ONG ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
16S Rdna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Gradient Gel Electrophoresis ◽  
Diversity Study ◽  
Rdna Clone

Survey of Oral Microbial Diversity using PCR-based Denaturing Gradient Gel Electrophoresis

2005 ◽  
Vol 84 (6) ◽  
pp. 559-564 ◽  
Author(s):  
Y. Li ◽  
C.Y.S. Ku ◽  
J. Xu ◽  
D. Saxena ◽  
P.W. Caufield
Keyword(s):  
Microbial Diversity ◽  
16S Rdna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Profile Analysis ◽  
Oral Bacteria ◽  
Clinical Samples ◽  
Computer Assisted ◽  
Oral Microbiota ◽  
Gradient Gel Electrophoresis

Polymicrobial biofilms in the human oral cavity exhibit marked diversity. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) surveys microbial diversity by displaying PCR-generated 16S rDNA fragments that migrate at different distances, reflecting the differences in the base-pair ( i.e., % G+C) composition of the fragment. This study examined DGGE-generated diversity profiles of cultivable bacteria from individuals with different caries status. Initially, we developed a set of PCR-DGGE running conditions appropriate to oral bacteria. Next, we assessed migration standards from known oral bacterial reference strains. To test the methods, we profiled 20 bacterial saliva samples cultivated from young adults. The study produced a battery of species-specific 16S rDNA amplicons that could be used as a migration distance standard necessary for computer-assisted profile analysis. From the clinical samples, we found a significantly greater diversity of oral microbes in caries-free individuals compared with caries-active individuals ( P = 0.01). These findings suggest thtat a portion of oral microbiota of caries-active individuals may be absent, suppressed, or replaced.

Isolation and purification of microbial community DNA from soil naturally enriched for chitin

Biologia ◽  
2012 ◽  
Vol 67 (4) ◽  
Author(s):  
Pullabhotla Sarma ◽  
Vadlamudi Srinivas ◽  
Kondreddy Anil ◽  
Appa Podile
Keyword(s):  
16S Rdna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Sodium Borate ◽  
Metagenomic Dna ◽  
Soil Dna ◽  
Isolation And Purification ◽  
Gradient Gel Electrophoresis ◽  
Chitinase Genes ◽  
Sodium Borate Buffer

AbstractWe made an attempt to isolate and purify metagenomic DNA from chitin enriched soil. In this communication we report a modified direct lysis method for soil DNA extraction including initial pre-lysis washing of sample, followed by a rapid polyvinylpyrrolidone-agarose-based purification and electroelution of DNA using Gene-capsule™ assembly. Rapidity was achieved using low molarity conducting media (sodium-borate buffer) for electrophoresis by reducing run time for both the gel electrophoresis and electroelution. Extracted DNA was sufficiently pure and of high quality, evidenced by amplification of 16S rDNA and chitinase genes by PCR. Metagenomic nature of the DNA was confirmed by running V3 (16S rDNA) region amplicons using denaturing gradient gel electrophoresis. This method requires 30 min for purification, and less than 2 h for complete execution of protocol and becomes the first report on the isolation of metagenomic DNA from soil naturally enriched for chitin.

scholarly journals Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

2003 ◽  
Vol 69 (11) ◽  
pp. 6801-6807 ◽  
Author(s):  
Isabel Lopez ◽  
Fernanda Ruiz-Larrea ◽  
Luca Cocolin ◽  
Erica Orr ◽  
Trevor Phister ◽  
...  
Keyword(s):  
Lactic Acid ◽  
16S Rdna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Species ◽  
Pcr Primers ◽  
Fungal Species ◽  
Bacterial Populations ◽  
Gradient Gel Electrophoresis ◽  
Primer Sets

ABSTRACT Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.

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