• The Plant Pathology Journal
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• v.26 no.3
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• pp.289-292
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• 2010

This study used a modified cetyltrimethylammonium bromide (CTAB) method to efficiently extract DNA from the plant pathogenic fungus Cercospora sojina. Total DNA yield obtained by this method was approximately 1 mg/g of mycelia (fresh weight), and the mean ratio of A260/A280 and A260/A230 were 2.04 and 2.1, respectively. The results of random amplified polymorphic DNA (RAPD) analysis, digestion with restriction enzymes, and Southern hybridization indicated that polysaccharides were effectively removed by this method, and the resulting DNA was sufficient for use in subsequent molecular analysis.

• Korean Journal of Ecology and Environment
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• v.54 no.3
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• pp.170-189
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• 2021

Environmental DNA (eDNA) is a genetic material derived from organisms in various environments (water, soil, and air). eDNA has many advantages, such as high sensitivity, short investigation time, investigation safety, and accurate species identification. For this reason, it is used in various fields, such as biological monitoring and searching for harmful and endangered organisms. To collect eDNA from a freshwater ecosystem, it is necessary to consider the target organism and gene and a wide variety of items, such as on-site filtration and eDNA preservation methods. In particular, the method of collecting eDNA from the environment is directly related to the eDNA concentration, and when collecting eDNA using an appropriate collection method, accurate (good quality) analysis results can be obtained. In addition, in preserving and extracting eDNA collected from the freshwater ecosystem, when an accurate method is used, the concentration of eDNA distributed in the field can be accurately analyzed. Therefore, for researchers at the initial stage of eDNA research, the eDNA technology poses a difficult barrier to overcome. Thus, basic knowledge of eDNA surveys is necessary. In this study, we introduced sampling of eDNA and transport of sampled eDNA in aquatic ecosystems and extraction methods for eDNA in the laboratory. In addition, we introduced simpler and more efficient eDNA collection tools. On this basis, we hope that the eDNA technique could be more widely used to study aquatic ecosystems and help researchers who are starting to use the eDNA technique.

• Analytical Science and Technology
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• v.31 no.2
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• pp.65-70
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• 2018

This study compared two methods for preparing ancient DNA (aDNA) for the construction of successful shotgun libraries that may be applied to massive parallel sequencing. For the comparative analysis, the DNA of prehistoric rib samples from Hungary was extracted using either a manually prepared silica suspension or the Amicon Ultracel-15 10K ultracentrifugal device (Millipore). After the extraction of the same amount of bone powder (about 150 mg) from three samples by each method, the amount of extracted double-stranded DNA and the subsequent degree of construction of the shotgun library were analyzed. The Amicon device method was rapid and easier to perform and resulted in an approximately 11-fold higher DNA recovery than that obtained using the silica suspension. The shotgun library constructed using DNA templates prepared by the Amicon device was more successful than that constructed from templates isolated using the silica suspension. The comparative study of these two aDNA extraction methods showed that the Amicon device has the advantages of saving time, process simplicity, and high efficiency.

• Journal of the korean academy of Pediatric Dentistry
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• v.41 no.4
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• pp.292-297
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• 2014

DNA extraction is a prerequisite for the identification of pathogens in clinical samples. Commercial DNA extraction kits generally involve time-consuming and laborious multi-step procedures. In the present study, our modified DNA isolation method for saliva samples allows for the quick detection of pathogens associated with dental caries or periodontitis by PCR within 1 h. To release DNA from the bacteria, 1 min of boiling was adequate, and the resulting isolated DNA can be used many times and is suitable for long term storage of at least 13 months at $4^{\circ}C$, and even longer at $-20^{\circ}C$. In conclusion, our modified DNA extraction method is simple, rapid, and cost-effective, and suitable for preparing DNA from clinical samples for PCR for the rapid detection of oral pathogens from saliva.

• Journal of Microbiology and Biotechnology
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• v.24 no.6
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• pp.812-822
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• 2014

Metagenomic analysis of the human intestinal microbiota has extended our understanding of the role of these bacteria in improving human intestinal health; however, a number of reports have shown that current total fecal DNA extraction methods and 16S rRNA universal primer sets could affect the species coverage and resolution of these analyses. Here, we improved the extraction method for total DNA from human fecal samples by optimization of the lysis buffer, boiling time (10 min), and bead-beating time (0 min). In addition, we developed a new long-range 16S rRNA universal PCR primer set targeting the V6 to V9 regions with a 580 bp DNA product length. This new 16S rRNA primer set was evaluated by comparison with two previously developed 16S rRNA universal primer sets and showed high species coverage and resolution. The optimized total fecal DNA extraction method and newly designed long-range 16S rRNA universal primer set will be useful for the highly accurate metagenomic analysis of adult and infant intestinal microbiota with minimization of any bias.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.284-289
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• 2004

A PCR assay for the rapid detection of Vibrio vulnificus strains was developed using a virulence gene for elastase found in various Vibrio species. The DNA sequences in the elastase gene facilitated the identification of a species-specific probe for pathogenic V. vulnificus strains from both clinical and environmental sources. Using an elastase gene-based PCR reaction, a species-specific 507-bp PCR product was visualized by agarose gel electrophoresis. Three different DNA extraction methods were then compared to improve the simplicity and rapidity of detection. A PCR assay using the conventional DNA extraction or boiling method was able to detect as few as 25 V. vulnificus cells, making the detection limits at least 1-log-scale lower than that for the EDT A-treated DNA extraction method. In particular, the boiling method, which does not require purification of the chromosomal DNA, was very effective in terms of simple and rapid detection. Meanwhile, the detection limit in a mixed bacterial culture that included other bacteria, such as Escherichia coli or Bacillus subtilis, was two V. vulnificus cells, which was 1-log-scale lower than that for the control. Accordingly, when coupled with a new DNA extraction method, the elastase gene-based PCR can provide a rapid, specific, and sensitive method for identifying V. vulnificus in clinical and environmental samples.

• Journal of Biomedical Engineering Research
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• v.30 no.4
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• pp.311-317
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• 2009

An automatic control system is proposed and implemented for a miniaturized DNA extraction system using magnetic bead. A host-local system is employed for the accommodation of the graphical user interface and the basic control function. The functional partitioning into the local and the host system is discussed. The control functions are classified and formalized for the flexible control scenario, which is the input of the proposed system. As the proposed scenario is consists of the sequence of the user-centric actions, the user goal can be easily programmed and modified. The DNA extraction performance of the implemented system was compared with the existing silica-membrane-based method, resulting in the comparable concentration and purity of the extracted DNA. The proposed system is currently being utilized for the development of the DNA extraction system only changing scenario, without any alteration of the system.

• Plant Biotechnology Reports
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• v.4 no.1
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• pp.49-52
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• 2010

A DNA extraction method using Chelex 100 is widely used for bacteria, Chlamydomonas, and animal cell lines, but only rarely for plant materials due to the need for additional time-consuming and tedious steps. We have modified the Chelex 100 protocol and successfully developed a rapid and simple method of DNA extraction for efficient PCR-based detection of transgenes from a variety of transgenic plant and algal species. Our protocol consists of homogenizing plant tissue with a pestle, boiling the homogenized tissue in a microfuge tube with 5% Chelex 100 for 5 min, and centrifuging the boiled mixture. The supernatant, which is used for PCR analysis, was able to successfully amplify transgenes in transgenic tobacco, tomato, potato, Arabidopsis, rice, strawberry, Spirodela polyrhiza, Chlamydomonas, and Porphyra tenera. The entire DNA extraction procedure requires <15 min and is therefore comparable to that used for bacteria, Chlamydomonas, and animal cell lines.

• Korean Journal of Food Science and Technology
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• v.48 no.4
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• pp.335-340
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• 2016

This study was conducted to find out the most suitable DNA isolation methods for PCR detection of foodborne pathogens. Four DNA isolation methods including Universal Genomic DNA Extraction Kit (TaKaRa), PrepMan Ultra (Applied Biosystems), boiling method and alkaline lysis method (w/PEG) were tested and compared. The Universal Genomic DNA Extraction kit (TaKaRa) was considered as the more efficient isolation method for Escherichia coli O157:H7 and Staphylococcus aureus in lettuce, fish and beef. Meanwhile to detect the foodborne pathogens directly from foods without enrichment, the four different buffers such as double-distilled water, saline, glycine-saline, glycine-saline with Tween-20 and beef extract were also evaluated. As a result, saline was more suitable buffer for E. coli O157:H7. And double-distilled water was more suitable buffer than saline for S. aureus, respectively

• Mycobiology
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• v.42 no.4
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• pp.311-316
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• 2014

Lichen studies, including biodiversity, phylogenetic relationships, and conservation concerns require definitive species identification, however many lichens can be challenging to identify at the species level. Molecular techniques have shown efficacy in discriminating among lichen taxa, however, obtaining genomic DNA from herbarium and fresh lichen thalli by conventional methods has been difficult, because lichens contain high proteins, polysaccharides, and other complex compounds in their cell walls. Here we report a rapid, easy, and inexpensive protocol for extracting PCR-quality DNA from various lichen species. This method involves the following two steps: first, cell breakage using a beadbeater; and second, extraction, isolation, and precipitation of genomic DNA. The procedure requires approximately 10 mg of lichen thalli and can be completed within 20 min. The obtained DNAs were of sufficient quality and quantity to amplify the internal transcribed spacer region from the fungal and algal lichen components, as well as to sequence the amplified products. In addition, 26 different lichen taxa were tested, resulting in successful PCR products. The results of this study validated the experimental protocols, and clearly demonstrated the efficacy and value of our KCl extraction method applied in the fungal and algal samples.