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| Optimization of Denaturing Gradient Gel Electrophoresis (DGGE) Protocol in Analyzing Rumen Bacterial Community Diversity |
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Abstract In order to improve the accuracy of denaturing gradient gel electrophoresis (DGGE) in the analysis of rumen bacteria community diversity, the best range of DGGE denaturing gradient, PCR amplification procedure and treatments methods of PCR products had been optimized in this study. Three denaturing gradients, 35%~70%, 40%~60% and 55%~65%, were designed for DGGE, and normal PCR and reconditioning PCR were used to amplify rumen bacteria 16S rDNA, respectively. After that, the PCR products were purified via denatured polyacrylamide gel electrophoresis (d-PAGE) and mung bean nuclease respectively, as well as cluster and some bands sequences analysis. The results showed that the DNA bands in gel were well-distributed at 40%~60% denaturing gradients. Reconditioning PCR plus d-PAGE purification could minimize single-stranded DNA (ssDNA) to a certain extent. According to cluster and sequence analysis, different treatments method could result in different bands distribution in DGGE profiles, and the single strand could not completely represented unique bacteria species. In conclusion, the better denaturing gradient for rumen bacteria community DGGE was 40%~60%, and reconditioning PCR plus d-PAGE purification could be used to purify samples before DGGE analysis to obtain the better profile. The study provides basic references to DGGE in the analyzing of rumen bacteria community diversity.
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Received: 21 October 2014
Published: 13 April 2015
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