• Proceedings of the Korean Aquaculture Society Conference
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• 2003.10a
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• pp.134-135
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• 2003

The geographic expansion of the toxic dinoflagellates genus Alexandrium has been shown to be world wide ranging. The members of the genus Alexandrium ocnstituted of 20-30 species did not show substantial differences in their morphology, which is mostly referred in the 'tamarensis species complex', except some species. Though rDNA sequences variations are very few and pseudogene types are so diverse that it is difficult to use them as the specific markers. In this study, we outlined Korean and Japanese A, tamarense and A. catenella regional isolates by phylogenetic analysis inferred from no cutting alignments of LSU rDNA D1-D2 and SSU rDNA sequences to group these regional isolates. The results were compared to RFLP patterns of PCR products targeted chloroplast DNA. Lastly screening of highly repeated microsatellite DNA which is frequently used for population analysis in eukaryotes was conducted. A. catenella regional strains identified by the sequencing of rDNA D1-D2 domain were divided into at least 3 groups of type E, CMC and Chinese type, divergence root may not be deep comparing with that of A. tamarense whose pseudogenes are very variable. Results of RFLP pattern and the phylogeny of the unknown gene targeting chloroplast showed that Korean and Japanese A. catenella regional isolates were divided into 3 types: Korean, Japanese and the third CMC types. Population-specific PCR amplification with Japanese A. catenella type-specific PCR primers was useful method for population analysis of A. catenella. Various types of satellite sequences such as 5 nucleotides repeats were obtained from A. tamarense and A. catenella. The 5 nucleotides repeats were primed at the both 3'and 5' ends, and these repeats were prominent as longer repeated motifs. This repeated DNA was intercalated as internal sequences containing various types subrepeats. It is expected that these satellite DNA would be a useful molecular population marker through detail comparison among Alexandrium regional isolates to trace their transferring pathway and to prevent their human-associated their regional extents.

• Journal of Korean Society of Forest Science
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• v.96 no.4
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• pp.425-431
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• 2007

To investigate genetic diversity and PCR detection of Rhizina undulata, PCR detection and sequence analysis of rDNA ITS region of R. undulata in soil were analyzed and developed. The length of partial 18S rDNA from four R. undulata isolates were 1,375 nt. The sequence similarity of R. undulata isolates was 100%. The rDNA ITS regions of R. undulata isolates were 585 nt long. Nucleotide sequencing of the ITS regions showed that PDK-1, PTT-1 and PDJ-9 isolates had 100% sequence identity. But, PDS-5 isolate differed from the three isolates by two nucleotide substitution. R. undulata-specific primers designed by the sequence of ITS region were used in PCR detection of R. undulata. PCR products about 525 bp size, which is specific to R. undulata, were amplified from total DNAs of R. undulata isolates. To assay the sensitivity of PCR detection by R. undulata ITS-specific primer, purely cultured mycelial suspension of R. undulata was serially diluted and mixed with 100g of sterile sandy loam soil, respectively. And then, PCR products of total DNAs extracted from each mycelium-soil mixtures were analysed. The PCR protocol could detected up to 1ng mycelium of R. undulata within 100g of soil.

• Biomedical Science Letters
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• v.2 no.2
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• pp.223-229
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• 1996

HLA-B27 gene, one of the HLA-class I molecule, is strongly associated with ankylosing spondylitis. It has been most frequently used as a disease-correlated HLA gene by clinicians. In most laboratories, conventional HLA-B27 typing is still performed by cell cytotoxicity tests or fluorescence serology with specific antibodies. In this study, DNA typing method for HLA-B27 was developed by using group specific Polymerase Chain Reaction (PCR). Four HLA-B27 cell lines (HOM-2, JESTHOM, WT24 and BTB) and fifty six B27 Korean individuals defined by serology were used. The results of control cell and B-27 positive individual samples were correlated well with the data which was performed by serological method. All of B27 positive PCR products gave positive signals on Southern blot hybridization with B27 specific probe. This study shows that the HLA-B27 DNA typing is a relatively simple, fast and practical tool for the determination of the HLA-B27 gene in routine clinical laboratory work.

• Animal cells and systems
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• v.9 no.2
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• pp.79-85
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• 2005

Oxidized abasic residues arise as a major class of DNA damage by a variety of agents involving free radical attack and oxidation of deoxyribose sugar components. 2-deoxyribonolactone (dL) is a C1'-oxidized abasic lesion implicated in DNA strand scission, mutagenesis, and covalent DNA-protein cross-link (DPC). We show here that mammalian cell-free extract give rise to stable DPC formation that is specifically mediated by dL residue. When a duplex DNA containing dL at the site-specific position was incubated with cell-free extracts of Po ${\beta}-proficient$ and -deficient mouse embryonic fibroblast cells, the formation of major dL-mediated DPC was dependent on the presence of DNA polymerase (Pol) ${\beta}$. Formation of dL-specific DPC was also observed with histones and FEN1 nuclease, although the reactivity in forming dL-mediated DPC was significantly higher with Pol ${\beta}$ than with histones or FEN1. DNA repair assay with a defined DPC revealed that the dL lesion once cross-linked with Pol ${\beta}$ was resistant to nucleotide excision repair activity of cell-free extract. Analysis of nucleotide excision repair utilizing a model DNA substrate containing a (6-4) photoproduct suggested that excision process for DPC was inhibited because of DNA single-strand incision at 5' of the lesion. Consequently DPC mediated by dL lesion may not be readily repaired by DNA excision repair pathway but instead function as unusual DNA damage causing a prolonged DNA strand break and trapping of the major base excision repair enzyme.

• Journal of Microbiology and Biotechnology
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• v.20 no.10
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• pp.1463-1470
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• 2010

E. coli has long been widely used as a host system for the manufacture of recombinant proteins intended for human therapeutic use. When considering the impurities to be eliminated during the downstream process, residual host cell DNA is a major safety concern. The presence of residual E. coli host cell DNA in the final products is typically determined using a conventional slot blot hybridization assay or total DNA Threshold assay. However, both the former and latter methods are time consuming, expensive, and relatively insensitive. This study thus attempted to develop a more sensitive real-time PCR assay for the specific detection of residual E. coli DNA. This novel method was then compared with the slot blot hybridization assay and total DNA Threshold assay in order to determine its effectiveness and overall capabilities. The novel approach involved the selection of a specific primer pair for amplification of the E. coli 16S rRNA gene in an effort to improve sensitivity, whereas the E. coli host cell DNA quantification took place through the use of SYBR Green I. The detection limit of the real-time PCR assay, under these optimized conditions, was calculated to be 0.042 pg genomic DNA, which was much higher than those of both the slot blot hybridization assay and total DNA Threshold assay, where the detection limits were 2.42 and 3.73 pg genomic DNA, respectively. Hence, the real-time PCR assay can be said to be more reproducible, more accurate, and more precise than either the slot blot hybridization assay or total DNA Threshold assay. The real-time PCR assay may thus be a promising new tool for the quantitative detection and clearance validation of residual E. coli host cell DNA during the manufacturingprocess for recombinant therapeutics.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.186-191
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• 2002

The usefulness of oligonucleotide DNA chip was evaluated for detection and primary level identification of major waterborne viruses in environmental samples. The enteric waterborne viruses included enterovirus, adenovirus, and rotavirus. Total intracellular RNA of 10 BGM cell plates showing virus-specific cytopathic effects was extracted at the third day after inoculation. The intracellular RNA was then subjected to either enterovirus-specific RT-PCR followed by sequencing analysis, or the DNA chip. Seven out of 10 positive samples in cell culture were positive but the other three sample were turned out to be negative by both RT-PCR and DNA chip analyses. Nucleotide sequencing results and the DNA chip hybridization results of the RT-PCR product were in complete agreement in the identification of the 7 positive samples as enteroviruses. Using the DNA chip, it took only 3∼4 hr to complete detection and primary level identification of target viruses and additional procedures such as gel electrophoresis or nucleotide sequencing were not necessary. We believe that the DNA chip system can be employed as a highly effective and new detection methodology for environmental viruses.

• Fisheries and Aquatic Sciences
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• v.7 no.3
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• pp.130-135
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• 2004

The marine dinoflagellate genus Alexandrium, which produces PSP toxins, has a global distribution. As human-assisted dispersal of the species has been suggested, it is important to develop molecular tools to trace the dispersal pathway. To screen population-specific DNA sequences that differentiate Korean and Japanese A. catenella, we targeted the area downstream of the chloroplast psbA gene using PCR with population-specific DNA primers followed by RFLP (restriction fragment length polymorphism) analysis and sequencing. The RFLP patterns of the PCR products divided Korean and Japanese A. catenella regional isolates into three types: Korean, Japanese, and type CMC3, isolated from Korea. We sequenced the PCR products, but found no similar gene in a homology search. The molecular phylogeny inferred from the sequences separated the Korean and Japanese A. catenella strains, as did the RFLP patterns. However, the Japanese isolates included two slightly different sequences (types J and K), while the Korean sequence was the same as the Japanese K type. In addition, a unique sequence was found in the Korean strains CMC2 and CMC3. Population-specific PCR amplification with Japanese A. catenella type-specific PCR primers designed from the type J sequence yielded PCR products for Japanese strains only, showing that the unknown gene can be used for a population analysis of Korean and Japanese A. catenella.

• Fisheries and Aquatic Sciences
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• v.26 no.11
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• pp.678-688
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• 2023

Flatfish are one of the largest families in the order Pleuronectiformes and are economically important edible marine fish species. However, they have similar morphological characteristics leading to challenges in classifying correctly, which may result in mislabeling and illegal sales, such as fraudulent labeling of processed food. Therefore, accurate identification is important to ensure the quality and safety of domestic markets in Korea. Species-specific primers were prepared from the mainly consumed eleven species of the order Pleuronectiformes. To rapidly identify the 11 flatfish species, a highly efficient, rapid, multiplex polymerase chain reaction (PCR) with species-specific primers was developed. Species-specific primer sets were designed for the mitochondrial DNA cytochrome c oxidase subunit I gene. Species-specific multiplex PCR (MSS-PCR) either specifically amplified a PCR product of a unique size or failed. This MSS-PCR analysis is easy to perform and yields reliable results in less time than the previous Sanger sequencing methods. This technique could be a powerful tool for the identification of the 11 species b the family Pleuronectidae and can contribute to the prevention of falsified labeling and protection of consumer rights.

• Research in Plant Disease
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• v.20 no.1
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• pp.21-24
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• 2014

Clubroot caused by the obligate biotrophic protist Plasmodiophora brassicae Woronin is one of the most damaging diseases of Brassicaceae family. In this study, we developed species-specific primer sets for rapid and accurate detection of P. brassicae. The primer sets developed amplified a specific fragment only from P. brassicae DNA while they did not amplify a band from 10 other soilborne pathogens or from Kimchi cabbage. In sensitivity test, the species-specific primer set ITS1-1/ITS1-2 could work for approximately 10 spores/ml of genomic DNA showing more sensitivity and accuracy than previous methods. With quantitative real-time PCR test, the primer set detected less spores of P. brassicae than before, confirming that the species-specific primer set could be useful for rapid and accurate detection of P. brassicae.

• Development and Reproduction
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• v.15 no.4
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• pp.315-324
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• 2011

This study was conducted to detect the specific expressing genes by using RAPD-PCR and RFLP method in the Korea Native Brindled Cattle, Korean Native cow and Holstein cattle. And then, the specific marker gene was investigated by the analysis of the genes for detection significance according to the expressing pattern. We found the specific expression gene by the RAPD-PCR analysis in Korea Native Brindled Cattle. It was detected the differences of the species in the colour and external section. The Korea Native Brindled Cattle were vary low compare to the Korean Native cow and Holstein cattle by analysis result of polymorphism and distribution. And there were a found the specific marker gene by sequencing in the R9B gene fragment of Korea Native Brindled Cattle. And the sequencing result of the R9B was different between Korean Native cow and Holstein cattle. Thus, this gene can be apply as the specific marker gene in the Korea Native Brindled Cattle.