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| Optimization of DNA Extraction from Faeces for Molecular Biology Detection |
| 1, 1, 1,1, 1,韦露450103198306212562李丽婷120103198310162928 1 |
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Abstract Nowadays the importance of gut microbiota for the health of host has been more and more emphasized. The human microbiome project (HMP) has been launched, and researches on gut microbiota count for the most important contents of HMP. Studies on this field usually take means of molecular biology methods in which DNA extraction is the basis and key to get success of experiments. In order to explore the best protocol of extracting DNA from gut bacteria, we compared 5 common fecal genome extraction methods using rat (Rattus norvegicus) faeces as samples. Four aspects including DNA concentration, integrity, purity and diversity were evaluated. Finally, we optimized the best method in the dosage of faeces and times of differential centrifugation. The results showed that method of differential centrifugation, chemical and enzyme strategies and pheno-chlorofom extraction was more suitable in bacterial DNA extraction from rat faeces. And the optimizing experiment suggested that 0.2 g faeces and differential centrifugation once was suitable for gut microbiota metgenomic sequencing; 0.4 g faeces and differential centrifugation twice was suitable for detecting numbers and diversity of gut flora by 16S rRNA-PCR; 0.3 g faeces and differential centrifugation once was suitable for getting the least PCR inhibitors and the best DNA quality. Our research provides useful basic data for further study of gut microbiota.
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Received: 27 August 2013
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