| Title | Direct extraction of DNA from soil for amplification of 16S rRNA gene sequences by polymerase chain reaction |
|---|---|
| Author | Cho, Jae Chang · Lee, Dong Hun · Cho, Young Cheol · Cho, Jang Cheon · Kim, Sang Jong * |
| Address | Department of Microbiology, College of Natural Sciences, and Research Center for Molecular Microbiology, Seoul National University |
| Bibliography | Journal of Microbiology, 34(3),229-235, 1996, |
| DOI | |
| Key Words | Soil, DNA extraction, PCR |
| Abstract | Microgram quantities of DNA per gram soil were recovered with SDS- based and freeze-and thaw procedures. The average DNA fragment size was > 23 Kb. This method generated minimal shearing of extracted DNA. However, the DNA extracts still contained considerable amounts of humic impurities sufficient to inhibit PCR. Several approaches were used to reduce the interferences with the PCR (use of CTAF in extraction step, Elutip-d column purification, addition of BSA to PCR buffer) to accomplish PCR with DNA extract as a template. Most of the DNA extracts were not digested completely by restriction endonuclease, and CTAB-TREATED and Elutip-d column purified DNA extracts were partially digested. Regarding as restriction enzyme digestion, all PCRs failed to amplify 16S rRNA gene fragments in the DNA extracts. In the case of DNA extracts only where BSA was added to PCR buffer, PCR was successfully conducted whether the DNA extracts were treated with CTAB or purified with columns. However, these two treatments were indispensable for humic impurity-rich DNA extracts to generate the PCR-compatible DNA samples. Direct extraction of DNA, coupled with these procedures to remove and relieve interferences by humic impurities and followed by the PCR, can be rapid and simple method for molecular microbiological study on soil microorganisms. |
| Download PDF | Eng_340304_229-235p.pdf |