Comparison of Total DNA Extraction Methods for Microbial Community Form Polluted Soil
2015
Tanase, Ana-Maria | Mereuta, Ioana | Chiciudean, Iulia | Ionescu, Robertina | Milea, Ligia | Cornea, Calina Petruta | Vassu, Tatiana | Stoica, Ileana
DNA isolation represents the basic and probably the most important step in molecular biology for microbial strains, and even more, for microbial community analyses. Despite the development of molecular protocols for DNA microbial community isolation, there are still many drawbacks dependent of samples composition, and even the commercially available genomic isolation kits have significant limitations in recovering high genomic DNA amounts, especially from soil samples. Our study is aiming to compare and optimize a total microbial community DNA isolation protocol from polluted soil samples, estimating the amount and the purity of genomic DNA per g of soil, versus time requirements for each protocol, taking under consideration that our soil samples have a high content of humic acids. We checked several protocols for total DNA extraction, CTAB based, including a specific Kit for Soil DNA Isolation Kit NorgenBiotek. We estimated the time needed for each protocol, the amount of the DNA per gram of polluted soil, proteins and RNA contamination grade, by spectrophotometric analysis, but also the grade of PCR amplification inhibition. The most efficient method for our soil samples with high content of humic acid, suitable for further molecular analyses was the total DNA microbial community sample retrieved from Sagova et al. (2008) based protocol, with several adjustments. This protocol will be valuable for molecular analysis on microbial community profiling from environmental samples, especially from polluted soils.
显示更多 [+] 显示较少 [-]