Journal of Microbiology

Research Support, Non-U.S. Gov'ts

Enhanced method for microbial community DNA extraction and purification from agricultural yellow loess soil: Mathur Nadarajan Kathiravan , Geun Ho Gim , Jaewon Ryu , Pyung Il Kim , Chul Won Lee , Si Wouk Kim; J. Microbiol. 2015;53(11):767-775. Published online October 28, 2015; DOI: https://doi.org/10.1007/s12275-015-5454-0

Abstract: In this study, novel DNA extraction and purification methods were developed to obtain high-quantity and reliable quality DNA from the microbial community of agricultural yellow loess soil samples. The efficiencies of five different soil DNAextraction protocols were evaluated on the basis of DNA yield, quality and DNA shearing. Our suggested extraction
method
, which used CTAB, EDTA and cell membrane lytic enzymes in the extraction followed by DNA precipitation using isopropanol, yielded a maximum DNA content of 42.28 ± 5.59 μg/g soil. In addition, among the five different purification protocols, the acid-treated polyvinyl polypyrrolidone (PVPP) spin column purification method yielded high-quality DNA and recovered 91% of DNA from the crude DNA. Spectrophotometry revealed that the ultraviolet A260/A230 and A260/A280 absorbance ratios of the purified DNA were 1.82 ± 0.03 and 1.94 ± 0.05, respectively. PCR-based 16S rRNA amplification showed clear bands at ~1.5 kb with acid-treated PVPP–purified DNA templates. In conclusion, our suggested extraction and purification protocols can be used to recover high concentration, high purity, and high-molecular-weight DNA from clay and silica-rich agricultural soil samples.

NOTE] A Rapid PCR-Based Approach for Molecular Identification of Filamentous Fungi: Yuanyuan Chen , Bernard A. Prior , Guiyang Shi , Zhengxiang Wang; J. Microbiol. 2011;49(4):675-679. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0525-3

Abstract: In this study, a novel rapid and efficient DNA extraction method based on alkaline lysis, which can deal with a large number of filamentous fungal isolates in the same batch, was established. The filamentous fungal genomic DNA required only 20 min to prepare and can be directly used as a template for PCR amplification. The amplified internal transcribed spacer regions were easy to identify by analysis. The extracted DNA also can be used to amplify other protein-coding genes for fungal identification. This method can be used for rapid systematic identification of filamentous fungal isolates.

Direct extraction of DNA from soil for amplification of 16S rRNA gene sequences by polymerase chain reaction: Cho, Jae Chang , Lee, Dong Hun , Cho, Young Cheol , Cho, Jang Cheon , Kim, Sang Jong; J. Microbiol. 1996;34(3):229-235.

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.

Methods of the extraction of DNA from water samples for polymerase chain reaction: Jung, Hae Sung , Lee, Young Jong; J. Microbiol. 1997;35(4):354-359.

Abstract: Methods for the extraction of DNA from water sample were approximated. Four different procedures of DNA extraction were carried out with pellets obtained from centrifugation of 4 liter water samples. The recovery efficiency and purity of DNA extracted by each method from different sources were compared. DNA yield varied with extraction methods, Method I, which involves enzymatic and freeze-thaw lysis steps and phenol and phenol-chloroform purification of extracted nucleic acid, showed a significantly higher yield and purity than the other methods. The use of glass beads in the DNA extraction methods improved the purity of DNA suitable for PCR. Bovine serum albumin in the PCR reaction mixture was useful in reducing inhibitory effects of contaminants. The efficiency of an extraction method was determined by the detection of the aer of Aeromonas hydrophila with PCR. The lower limit of detection of A. hydrophila from seeded tap water was 2 CFU/ml in PCR when method I was used for DNA preparation.

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