Development of a fluorophore-ribosomal DNA restriction typing method for monitoring structural shifts of microbial communities |
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Authors: | Tingting Wang Xiaojun Zhang Menghui Zhang Linghua Wang Liping Zhao |
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Institution: | (1) Key Laboratory of Ministry of Education for Microbial Metabolism, School of Life Science & Biotechnology, Shanghai Jiao Tong University, 800 DongChuan Road, 200240 Shanghai, China;(2) Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 200240 Shanghai, China; |
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Abstract: | DNA restriction fragment polymorphism technologies such as amplified ribosomal DNA restriction analysis (ARDRA) and terminal
restriction fragment length polymorphism (T-RFLP) have been widely used in investigating microbial community structures. However,
these methods are limited due to either the low resolution or sensitivity. In this study, a fluorophore-ribosomal DNA restriction
typing (f-DRT) approach is developed for structural profiling of microbial communities. 16S rRNA genes are amplified from
the community DNA and digested by a single restriction enzyme Msp I. All restriction fragments are end-labeled with a fluorescent nucleotide Cy5-dCTP via a one-step extension reaction and
detected with an automated DNA sequencer. All 50 predicted restriction fragments between 100 and 600 bp were detected when
twelve single 16S rRNA gene sequences were analyzed using f-DRT approach; 92% of these fragments were determined with accuracy
of ±2 bp. In the defined model communities containing five components with different ratios, relative abundance of each component
was correctly revealed by this method. The f-DRT analysis also showed structural shifts of intestinal microbiota in carcinogen-treated
rats during the formation of precancerous lesions in the colon, as sensitive as multiple digestion-based T-RFLP analysis.
This study provides a labor and cost-saving new method for monitoring structural shifts of microbial communities. |
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