Improved purification and PCR amplification of DNA from environmental samples |
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Authors: | Arbeli Ziv Fuentes Cilia L |
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Affiliation: | Departamento de Agronomía, Facultad de Agronomía; Universidad Nacional de Colombia--Sede Bogotá, Bogotá, D.C., Colombia. aziv@unal.edu.co |
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Abstract: | Purification and PCR amplification procedures for DNA extracted from environmental samples (soil, compost, and river sediment) were improved by introducing three modifications: precipitation of DNA with 5% polyethylene glycol 8000 (PEG) and 0.6 M NaCl; filtration with a Sepharose 4B-polyvinylpolypyrrolidone (PVPP) spin column; and addition of skim milk (0.3% w/v) to the PCR reaction solution. Humic substances' concentration after precipitation with 5% PEG was 2.57-, 5.3-, and 78.9-fold lower than precipitation with 7.5% PEG, 10% PEG, and isopropanol, respectively. After PEG precipitation, Sepharose, PVPP and the combined (Sepharose-PVPP) column removed 92.3%, 89.5%, and 98%, respectively, of the remaining humic materials. Each of the above-mentioned modifications improved PCR amplification of the 16S rRNA gene. DNA extracted by the proposed protocol is cleaner than DNA extracted by a commercial kit. Nevertheless, the improvement of DNA purification did not improve the detection limit of atrazine degradation gene atzA. |
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Keywords: | environmental DNA DNA purification PCR inhibition polyethylene glycol 8000 Sepharose 4B–polyvinylpolypyrrolidone spin column skim milk |
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