AIM: To develop a simple and convenient method for extracting genomic DNA from intestinal microflora for en- terobacterial repetitive intergenic consensus (ERIC)-PCR detection. METHODS: Five methods of extracting bact...AIM: To develop a simple and convenient method for extracting genomic DNA from intestinal microflora for en- terobacterial repetitive intergenic consensus (ERIC)-PCR detection. METHODS: Five methods of extracting bacterial DNA, including Tris-EDTA buffer, chelex-100, ultrapure water, 2% sodium dodecyl sulfate and 10% Triton-100 with and without sonication, were compared with the commercial fecal DNA extraction kit method, which is considered as the gold standard for DNA extraction. The comparison was based on the yield and purity of DNA and the indexes of the structure and property of micro-organisms that were reflected by ERIC-PCR. RESULTS: The yield and purity of DNA obtained by the chelex method was similar to that obtained with the fecal DNA kit. The ERIC-PCR results obtained for the DNA extracted by the chelex method and those obtained for DNA extracted with the fecal DNA kit were basically the same.CONCLUSION: The chelex method is recommended for ERIC-PCR experiments in view of its simplicity and cost- effectiveness; and it is suitable for extracting total DNA from intestinal micro-organisms, particularly for handling a large number of samples.展开更多
The actinomycete populations and functions in cadmium （Cd） contaminated soil were investigated by the cultivation- independent molecular methods. The genomic DNA was extracted and purified from soil adulterated with...The actinomycete populations and functions in cadmium （Cd） contaminated soil were investigated by the cultivation- independent molecular methods. The genomic DNA was extracted and purified from soil adulterated with various con- centrations of Cd in the laboratory. The partial 16S rDNA genes were amplified by polymerase chain reaction （PCR） using specific primers bound to evolutionarily conserved regions within these actinomycete genes. The diversity in PCR- amplified products, as measured by denaturing gradient gel electrophoresis （EGGE）, was used as a genetic fingerprint of the population. Principle component analysis and Shannon-Weaver diversity index （H） analyses were used to analyze the DGGE results. Results showed that the two principal components accounted for only a low level of the total variance. The value H in contaminated soil was lower than that in the control at later stages of cultivation, whereas at earlier stages it was higher. Among the six sampling time points, the first, fifth and sixth weeks had the highest values of H. Significantly negative correlations between bioavallable Cd concentration and H values existed in the samples from weeks 2 （R = 0.929, P 〈 0.05） and 4 （R = 0.909, P 〈 0.05）. These results may shed light on the effect of Cd on the soil environment and the chemical behavior and toxicity of Cd to actinomycetes.展开更多
Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis. However, residual humic substances may remain with obtained environmental DNA, which int...Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis. However, residual humic substances may remain with obtained environmental DNA, which interferes downstream molecular analyses. To remedy this situation, two DNA extraction buffers （PIPES and Tris-HCl） and four purification strategies including our new modified low melting point gel purification method and three commercial kits from QIAEX, Omega and Promega were evaluated with diverse soil samples. The PIPES buffer （pH 6.5） is found to be more effective for removing the humic substances, but it leads to lower DNA yield and causes more severe DNA shearing than using the Tris-HC1 buffer （pH 8.0）. Gel purification and the Promega purification kit achieve much higher DNA recoveries than QIAEX or Omega kit, and higher purity of DNA is obtained by gel purification than by the Promega kit with both DNA extraction buffers mentioned above. Considering all results together, two alternative methods for DNA extraction and purification are proposed： one uses Tris-HCl buffer extraction and gel purification as the primary approach when the amount of soil or biomass is not a major concern, and the other uses PIPES buffer extraction and the Promega kit purification when severe DNA shearing and/or limited biomass occurs. Purified DNA samples by both methods are amenable for use as templates for whole community genome amplifications and PCR amplifications of bacterial 16S rRNA genes. It is demonstrated that these two alternative methods could be applied to a wide variety of environmental samples.展开更多
基金The National Science Technology Pillar Program, 2007Z06-017Program for New Century Outstanding Talents from Universities, NCET-04-0906/NCET-06-0818+1 种基金Sichuan Provincial Basic Research Program, 04JY029-006-1/04JY021-100/07JY029-017Program for Key Disciplines Construction of Sichuan Province, SZD0418
文摘AIM: To develop a simple and convenient method for extracting genomic DNA from intestinal microflora for en- terobacterial repetitive intergenic consensus (ERIC)-PCR detection. METHODS: Five methods of extracting bacterial DNA, including Tris-EDTA buffer, chelex-100, ultrapure water, 2% sodium dodecyl sulfate and 10% Triton-100 with and without sonication, were compared with the commercial fecal DNA extraction kit method, which is considered as the gold standard for DNA extraction. The comparison was based on the yield and purity of DNA and the indexes of the structure and property of micro-organisms that were reflected by ERIC-PCR. RESULTS: The yield and purity of DNA obtained by the chelex method was similar to that obtained with the fecal DNA kit. The ERIC-PCR results obtained for the DNA extracted by the chelex method and those obtained for DNA extracted with the fecal DNA kit were basically the same.CONCLUSION: The chelex method is recommended for ERIC-PCR experiments in view of its simplicity and cost- effectiveness; and it is suitable for extracting total DNA from intestinal micro-organisms, particularly for handling a large number of samples.
基金Project supported by the National Natural Science Foundation of China (Nos. 30570053 and 40501037)the National High Technology Research and Development Program (863 Program) of China (No. 2007AA10Z409)+1 种基金the National"Eleventh Five Years Plan" Key Project on Science and Technology of China (No. 2006BAJ08B01)the Research Fund of Science and Technology Bureau of Zhejiang Province,China (No. 2008C23088)
文摘The actinomycete populations and functions in cadmium （Cd） contaminated soil were investigated by the cultivation- independent molecular methods. The genomic DNA was extracted and purified from soil adulterated with various con- centrations of Cd in the laboratory. The partial 16S rDNA genes were amplified by polymerase chain reaction （PCR） using specific primers bound to evolutionarily conserved regions within these actinomycete genes. The diversity in PCR- amplified products, as measured by denaturing gradient gel electrophoresis （EGGE）, was used as a genetic fingerprint of the population. Principle component analysis and Shannon-Weaver diversity index （H） analyses were used to analyze the DGGE results. Results showed that the two principal components accounted for only a low level of the total variance. The value H in contaminated soil was lower than that in the control at later stages of cultivation, whereas at earlier stages it was higher. Among the six sampling time points, the first, fifth and sixth weeks had the highest values of H. Significantly negative correlations between bioavallable Cd concentration and H values existed in the samples from weeks 2 （R = 0.929, P 〈 0.05） and 4 （R = 0.909, P 〈 0.05）. These results may shed light on the effect of Cd on the soil environment and the chemical behavior and toxicity of Cd to actinomycetes.
基金Project(51104189)supported by the National Natural Science Foundation of ChinaProject(2010CB630901)supported by the National Basic Research Program of China+1 种基金Project(1343-77341)supported by the Graduate Education Innovative Program of Central South University,ChinaProject(DOE-ER64125)supported by Department of Energy,Office of Science under the Environmental Remediation Science Program of the United States
文摘Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis. However, residual humic substances may remain with obtained environmental DNA, which interferes downstream molecular analyses. To remedy this situation, two DNA extraction buffers （PIPES and Tris-HCl） and four purification strategies including our new modified low melting point gel purification method and three commercial kits from QIAEX, Omega and Promega were evaluated with diverse soil samples. The PIPES buffer （pH 6.5） is found to be more effective for removing the humic substances, but it leads to lower DNA yield and causes more severe DNA shearing than using the Tris-HC1 buffer （pH 8.0）. Gel purification and the Promega purification kit achieve much higher DNA recoveries than QIAEX or Omega kit, and higher purity of DNA is obtained by gel purification than by the Promega kit with both DNA extraction buffers mentioned above. Considering all results together, two alternative methods for DNA extraction and purification are proposed： one uses Tris-HCl buffer extraction and gel purification as the primary approach when the amount of soil or biomass is not a major concern, and the other uses PIPES buffer extraction and the Promega kit purification when severe DNA shearing and/or limited biomass occurs. Purified DNA samples by both methods are amenable for use as templates for whole community genome amplifications and PCR amplifications of bacterial 16S rRNA genes. It is demonstrated that these two alternative methods could be applied to a wide variety of environmental samples.
