The use of real-time PCR technology to assess the effectiveness of methods of DNA extraction from cultures of acidophilic chemolithotrophic microorganisms |
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| Authors: | S V Rogatykh A A Dokshukina T S Khainasova S V Muradov I A Kofiadi |
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| Institution: | 1.Research Geotechnological Center, Far Eastern Branch,Russian Academy of Sciences,Petropavlovsk-Kamchatsky,Russia;2.ZAO NPF DNA-Technology,Moscow,Russia |
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| Abstract: | Comparative evaluation of efficiency of several methods of DNA extraction from storage cultures of acidophilic chemolithotrophic
microorganism communities isolated from sulfide ores of Shanuch ore deposit (Kamchatka peninsula) was conducted. DNA extraction
methods in various combinations of physical (heating to 65–98°C, grinding with SiO2 particles), enzymatic (treatment with lysozyme and proteinase K), and chemical (GuSCN, CTAB and KOH) treatments were tested.
The evaluation of efficiency was performed using Real-time PCR. The best result was obtained for the combined method based
on GuSCN lysis activity (lysis at 65°C) followed by purification with phenol and chloroform. |
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