The Simultaneous Recovery And Separation Of Nucleic Acid From Mining Environmental Microbes

To tackle and use the low-grade ores as well as tailings difficult to deal with, such microbial technology as bioleaching and bioremediation is a feasiable, efficient, economic and environmentally-friendly method, which can be widely applied. In order to improve the efficacy of microorganism, we have to understand the construction of the communities, the mechanism of bioleaching and explore the high efficient microbes in such environments. At present, researchers have begun to find solution in genes using RFLP、Real-time PCR、gene chip or other advanced molecular technology, all of which are based on high quality DNA or RNA. Numerous as publics related to DNA or RNA extraction from various environmental samples are, however, the methods for co-recovery of DNA and RNA for these typical environmental samples are rarely reported. The aim of the research is to introduce an effective method for extraction and separation of DNA and RNA from these samples. Different pH values for extraction buffer and different amount of proteinase K were experimented; different salts, the optimized concentration of salt and nucleic acid as well as the separation conditions were determined. The results showed that the optimal situations for the extraction of DNA and RNA are as following:the pH of the PIPES buffer was7.0without the use of proteinase K.The amount of DNA is positive related to the amount of biomass (R=0.99), while the amount of RNA is not. This method can be applied in A.f, A.c, A.t, L.f, F.t as well as environmental samples. From biomass as much as109, we could gain DNA6.29±2.34μg, RNA14.15±2.36μg.Compared with NaCl,CaCl2,LiCl worked better in nucleic acid separation.1/4volume of saturated LiCl working on nucleic acid with concentration of400ng/μL could separate DNA from RNA by centrifugation after6-8h low temperature (4℃)settlement. The recovery rate of separated DNA and RNA was accepted. Meanwhile, the purity was high which could be proved by the accepted ratio of A260/280and A260/230and single peak at260nm. The DNA fragment greater than23kb and integrity16S and23S RNA were attained, which were qualified for the library construction, reverse transcript-PCR and other downstream molecular analysis, so the method can be applied in the microbial molecular research of samples from mining and milling environments and even other samples.