• 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.

• Tuberculosis and Respiratory Diseases
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• v.43 no.1
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• pp.30-37
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• 1996

Background: The extraction methods of DNA from clinical samples are the major obstacle to use the PCR(polymerase Chain Reaction) in routine labortary for early detection of M. tuberculosis. We tried to improve the extraction method of DNA from sputum for establishment of the PCR in routine labortary by reducing the possibility of cross contamination and performing it easily and safely. Methods: We used the $InstaGene^{TM}$ DNA extraction kit(BioRad Co.) using Chelex 100 ion exchange resin for preparation of DNA. We compared InstaGene method in 100 cases of sputum from proteinase K method which is known as the most commonly used method for DNA purification(Experiment 1). And we compared InstaGene method in 98 cases of sputum from Microwave method developed by a company in Korea(Experiment 2). In experiment 1,245bps of IS6110 were amplified and then 188bps were amplified by nested PCR. In experiment 2,536bps in primary PCR and 276bps in nested PCR were amplified and analysed by agarose gel electrophoresis and EtBr staining. Results: When we chose AFB smear, culture, or AFB smear and culture as a standard test, PCR had low specificity and positive predictive value in both experiments. The InstaGene method has higher value in sensitivity and negative predictive value significantly than proteinase K method. The InstaGene method and the Microwave methods were similar in sensitivity, specificity, positive predictive value and negative predictive value. Conclusion: Even though both methods had lower possibility of cross contamination, shorter time requirement, simplicity, and economic advantages than Proteinase K method, the InstaGene method was a little simpler than the Microwave method. Therefore, in terms of usefulness in clinical application, the Instagene method seems to be the most useful method in DNA extraction for detection of M. tuberculosis using PCR. The reliability of this method will be clarified by further studies with enough clinical samples.

• Journal of Oral Medicine and Pain
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• v.20 no.2
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• pp.515-528
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• 1995

The human genomic deoxyribonucleic acid(DNA) was extracted from the pulp, dentin of 22 teeth by clelex, phenol methods. Samples of the tooth-derived DNA were amplified by polymerase chain reaction(PCR), electrophosed for sex determination by detection of X-Y homologus amelogenin gene and D1S80 locus detection The following results have been achieved. 1. Chelex and phenol method are effective to sex determination in the pulp and dentin 2. Chelex method is not suitable for detection of D1S80 locus. 3. Concentration and purity of DNA for teeth using chelex method is lower than using phenol method. From the above investigation, chelex method is simple, rapid for sex determination, but it is not suitable for detection of VNTRs.

• Journal of Microbiology
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• v.34 no.3
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• pp.229-235
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• 1996

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 ane 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.

• Microbiology and Biotechnology Letters
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• v.51 no.1
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• pp.99-108
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• 2023

Several challenges arise in DNA extraction and gene amplification for airborne fungal metagenome analysis from a particulate matter (PM) samples. In this study, various conditions were tested to optimize the DNA extraction method from PM samples and polymerase chain reaction (PCR) conditions with primer set and annealing temperature. As a result of comparative evaluation of DNA extraction under various conditions, chemical cell lysis using buffer and proteinase K for 20 minutes and bead beating treatment were followed by using a commercial DNA extraction kit to efficiently extract DNA from the PM filter samples. To optimize the PCR conditions, PCR was performed using 10 primer sets for amplifying the ITS2 gene region. The concentration of the PCR amplicon was relatively high when the annealing temperature was 58℃ with the ITS3tagmix3/ITS4 primer set. Even under these conditions, when the concentration of the PCR product was low, nested PCR was performed using the primary PCR amplicon as the template DNA to amplify the ITS2 gene at a satisfactory concentration. Using the methods optimized in this study, DNA extraction and PCR were performed on 15 filter samples that collected PM2.5 in Seoul, and the ITS2 gene was successfully amplified in all samples. The optimized methods can be used for research on analyzing and interpreting the fungal metagenome of atmospheric PM samples.

• Journal of the Korean Wood Science and Technology
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• v.46 no.4
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• pp.402-414
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• 2018

Species identification of wood provides important information for archaeology, restoration of cultural assets, preventing illegal logging, and more. Wood species are usually identified based on their anatomical features with the use of a microscope. However, this method may not be able to distinguish between anatomically similar species or subspecies. To overcome this problem, wood species need to be identified at the molecular level using DNA sequencing. However, unlike living plant cells, wood is difficult to pulverize using a mortar, and DNA extraction from dried wood is challenging. To solve these problems, we propose a pretreatment method in which wood is pulverized using 60-grit sandpaper and hydrated with water for 2 days. Using this method, we were able to stably amplify the rpoB gene from the extracted DNA of Pinus rigida. In addition, sequence analysis of the rpoB gene revealed six single nucleotide polymorphisms (SNPs), which classified the rpoB sequences in the genus Pinus into five groups. Our data indicate that although these SNPs were not suitable for species identification, they can potentially be used to determine the origin of different wood subspecies or individual samples of wood.

• Korean Journal of Clinical Laboratory Science
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• v.46 no.3
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• pp.99-105
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• 2014

Isolating total DNA from small samples using traditional methods is difficult and inefficient mainly due to loss of DNA during filtration and precipitation. With advances in molecular pathology, DNA extraction from micro-dissected cells has become essential in handling clinical samples. Genomic DNA extraction using small numbers of cells can be very important to successfully PCR amplify DNA from small biopsy specimens. We compared our experimental genomic DNA extraction method (A) with two other commercially available methods: using spin columns (B), and conventional resins (C), and determined the efficacy of DNA extraction from small numbers of cells smeared on a glass slide. Approximately 50, 100, 200, 500 and 1000 cells were isolated from fine needle aspiration biopsy (FNAB) slides aspirated from histologically proven papillary thyroid carcinoma masses. DNA was extracted using the three techniques. After measuring DNA quantity, PCR amplification was performed to detect the ${\beta}$-globin and $BRAF^{V600E}$ gene mutations. DNA extracted by method (A) showed better yield than the other methods in all cell groups. With our method, a suitable amount of genomic DNA to produce amplification was extracted from as few as 50 cells, while more than 100 to 200 cells were required when methods (B) or (C) were applied. Our genomic DNA extraction method provides high quality and improved yields for molecular analysis. It will be especially useful for paucicellular clinical samples which molecular pathologists often confront when handling fine needle aspiration cytology, exfoliative cytology and small biopsy specimens.

• The Journal of Korean Obstetrics and Gynecology
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• v.25 no.2
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• pp.89-107
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• 2012

Objectives: This study was performed to develop therapeutic prescription that is more significant than existing ones through extraction method and formulation changes. Methods: Yukmijihwangtang(YMJHT) was extracted in 80% ethanol, and their relative anti-oxidant activities as well as anti-inflammatory effects through immune modulation were measured. Results: Both water and ethanol extracted YMJHT showed does-dependent DPPH elimination activities. ROS inhibition activity was greater in water extracted YMJHT except for Moutan Cortex. NO inhibition assay results indicated that all groups showed higher NO inhibition activities in RAW 264.7 cells in dose dependent manner. Water extracted group showed higher NO inhibition activity than that of ethanol extracted group. TNF-${\alpha}$ secretion inhibition assay using RAW 264.7 cells, water extracted YMJHT showed higher activity than ethanol extracts. Growth rate of spleen cells was greater in all tested groups, with higher rate in YMJHT-EtOH than YMJHT-DW. Suppression of gene expression of IFN-r in spleen cells stimulated by Con A was higher in YMJHT-EtOH than YMJHT-DW. Suppression of gene expression of IL-10 in spleen cells stimulated by Con A was highest in YMJHT-DW with 40%. Suppression of gene expression of IL-4 in spleen cells stimulated by Con A were significant with 90% or higher in all groups and that of IL-12p35 were also higher than 90% in all cases. Conclusions: From the results, it shows that YMJHT has anti-inflammatory effects through immune modulation. However, the difference between YMJHT-EtOH and YMJHT-DW was not that significant. Further studies are needed to find out effective extraction methods of herbal medicine.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.3
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• pp.183-190
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• 2001

Objective: Recently, microdissection of tissue sections has been used increasingly for the isolation of morphologically identified homogeneous cell populations, thus overcoming the obstacle of tissue complexity for the analysis cell-specific expression of macromolecules. The aim of the present study was to establish the minimal conditions required for the RNA extraction and amplification from the cells captured by the laser captured microdissection. Methods : Mouse ovaries were fixed and cut into serial sections (7 im thickness). Oocytes were captured by laser captured microdissection (LCM) method by using PixCell $II^{TM}$ system. The frozen sections were fixed in 70% ethanol and stained with hematoxylin and eosin, while the paraffin sections were stained with Multiple stain. Sections were dehydrated in graded alcohols followed by xylene and air-dried for 20 min prior to LCM. All reactions were performed in ribonuclease free solutions to prevent RNA degradation. After LCM, total RNA extraction from the captured oocytes was performed using the guanidinium isothiocyanate (GITC) solution, and subsequently evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Results: With the frozen sections, detection of the GAPDH mRNA expression in the number of captured 25 oocytes were not repeatable, but the expression was always detectable from 50 oocytes. With 25 oocytes, at least 27 PCR cycles were required, whereas with 50 oocytes, 21 cycles were enough to detect GA PDH expression. Amount of the primary cDNA required for RT-PCR was reduced down to at least 0.25 $\grave{i}$ l with 50 oocytes, thus the resting 19.75 il cDNA can be used for the testing other interested gene expression. Tissue-to-slide, tissue-to-tissue forces were very high in the paraffin sections, thus the greater number of cell procurement was required than the frozen sections. Conclusion: We have described a method for analyzing gene expression at the RNA level with the homogeneously microdissected cells from the small amount of tissues with complexity. We found that LCM coupled with RT-PCR could detect housekeeping gene expression in 50 oocytes captured. This technique can be easily applied for the study of gene expression with the small amount of tissues.

• Animal cells and systems
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• v.9 no.4
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• pp.205-209
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• 2005

A rapid, simple and efficient method to extract RNA from the adult polyps of a soft coral, marine cnidarian, Scleronephthya gracillimum $(K\ddot{u}kenthal)$; was developed in this study. The highest yield and purity of RNA was obtained with the lysis solution containing 35 mM EDTA, 0.7 M LiCl, 7.0% SDS, and 200 mM Tris-Cl (pH 9.0). Approximately $40{\mu}g$ of total RNA was extracted from 200 mg of liquid nitrogen-pulverized polyp tissue. The ratio of absorbance at 260 nm and 280 nm ranged from 1.8 to 2.0. The results of the reverse transcription polymerase chain reaction (RTPCR) with ${\beta}-actin$ gene specific primers and Northern blot analysis using the same gene probe revealed that the RNA extracted by our method had high quality, and was sufficient for subsequent molecular biological analyses. This method was effective for RNA extraction from other soft coral species which belong to the genus Dendronephthya.