• Journal of Microbiology and Biotechnology
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• v.25 no.9
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• pp.1401-1409
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• 2015

The aim of this study was to develop a SYBR Green-based real-time PCR assay for the rapid, specific, and sensitive detection of Acidovorax avenae subsp. citrulli, which causes bacterial fruit blotch (BFB), a serious disease of cucurbit plants. The molecular and serological methods currently available for the detection of this pathogen are insufficiently sensitive and specific. Thus, a novel SYBR Green-based real-time PCR assay targeting the YD-repeat protein gene of A. avenae subsp. citrulli was developed. The specificity of the primer set was evaluated using DNA purified from 6 isolates of A. avenae subsp. citrulli, 7 other Acidovorax species, and 22 of non-targeted strains, including pathogens and non-pathogens. The AC158F/R primer set amplified a single band of the expected size from genomic DNA obtained from the A. avenae subsp. citrulli strains but not from the genomic DNA of other Acidovorax species, including that of other bacterial genera. Using this assay, it was possible to detect at least one genomeequivalents of the cloned amplified target DNA using 5 × 10⁰ fg/µl of purified genomic DNA per reaction or using a calibrated cell suspension, with 6.5 colony-forming units per reaction being employed. In addition, this assay is a highly sensitive and reliable method for identifying and quantifying the target pathogen in infected samples that does not require DNA extraction. Therefore, we suggest that this approach is suitable for the rapid and efficient diagnosis of A. avenae subsp. citrulli contaminations of seed lots and plants.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.819-824
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• 2001

The principal DNA modification systems of the amino-acid-producing bacteria Corynebacterium glutamicum AS019, Brevibacterium flavum BF4, and B. lactofermentum BL1 was investigated using two approaches; digestion of plasmid DNA isolated from these species TseI and Fnu4HI, and sequence analysis of the putative methyltransferase target sites following the derivatization of DNA using metabisulfite treatment. The C. glutamicum and B. flavum strains showed similar digestion patterns to the two enzymes, indicating that the target for cytidine methyltransferase recognizes 5'-GCSGC-3'(where S is either G or C). Mapping the methylated cytidine sites by bisulfite derivatization, followed by PCR amplification and sequencing, was only possible when the protocol included an additional step eliminating any underivatized DNA after PCR amplification, thereby indicating that the derivatization was not $100\%$ efficient. This may have been due to the high G0C content of this genus. It was confirmed that C. glutamicum AS019 and B. flavum BF4 methylated the cytidine in the $Gm^5CCGC$ sequences, yet there were no similar patterns of methylation in B. lactofermentum, which was consistent with the distinctive degradation pattern seen for the above enzymes. These findings demonstrate the successful application of a modified bisulfite derivatization method with the Corynebacterium species for determining methylation patterns, and showed that different species in the geneus contain distinctive restriction and modification systems.

• Journal of Life Science
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• v.24 no.4
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• pp.428-436
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• 2014

Loop-mediated isothermal amplification (LAMP) technique relies on autocycling strand displacement DNA sysnthesis without template denaturation steps under isothermal conditions. LAMP has more advantages than modern PCR, as it requires only basic laboratory equipment like an isothermal water bath, oven, and heating cabinet. Hence, in this study, five random primers were designed with Streptococcus parauberis, shikimate kinase Arok gene sequences (Genbank accession number: CP0024711). Two primers were selected based on the ladder pattern. Other optimum reaction conditions like temperature, time, and sensitivity were established and confirmed with conventional SYBR-green PCR. Results confirmed that the designed random primers were species specific, without any non-target DNA amplification under isothermal conditions. Hence, this study suggests that LAMP techniques could be used in the diagnosis of fish pathogen, specifically S. parauberis.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.5
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• pp.349-356
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• 2009

We previously reported that glial cell line-derived neurotropic factor (GDNF) receptor ${\alpha}$ 1 (GFR ${\alpha}$ 1) is a direct target of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1). In the present study, we further analyzed the physiological roles of Ape1/Ref-1-induced GFR ${\alpha}$ 1 expression in Neuro2a mouse neuroblastoma cells. Ape1/Ref-1 expression caused the clustering of GFR ${\alpha}$ 1 immunoreactivity in lipid rafts in response to GDNF. We also found that Ret, a downstream target of GFR ${\alpha}$ 1, was functionally activated by GDNF in Ape1/Ref-1-expressing cells. Moreover, GDNF promoted the proliferation of Ape1/Ref-1-expressing Neuro2a cells. Furthermore, GFR ${\alpha}$ 1-specific RNA experiments demonstrated that the downregulation of GFR ${\alpha}$ 1 by siRNA in Ape1/Ref-1-expressing cells impaired the ability of GDNF to phosphorylate Akt and PLC ${\gamma}$-1 and to stimulate cellular proliferation. These results show an association between Ape1/Ref-1 and GDNF/GFR ${\alpha}$ signaling, and suggest a potential molecular mechanism for the involvement of Ape1/Ref-1 in neuronal proliferation.

• Proceedings of the KIEE Conference
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• 2002.11a
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• pp.260-262
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• 2002

The determination of DNA hybridization can apply the molecular biology research. clinic diagnostics. bioengineering, environment monitoring, food science and other application area. So, The determination of hybridization is very important for the improvement of DNA detection system. In this study, we report the characterization of the DNA hybridization by the electricalchemical methods. The probe oligonucleotide was used to determine the amount of target oligonucleotide in solution using Methylen Blue(MB) as the electrochemical indicators. The cathodic peak currents($I_{peak}$) of MB were linearly related to the concentration of the target oligonucleotide sequence in the range $1[{\mu}M]{\sim}0.1[{\mu}M]$. The detection limit of this approach was 0.01[nM]. As a result, the match oligonucleotide(CR-1) was most stable state and the peak of redox current measured by DNA hybridization detection sensors by using electrochemical method seem to be similar to 1-mer terminal mismatch oligonucleotide(MR-3). The MR-2, MR-3, MR-22 and MR-33 have each mismatching sequence of central and terminal. With this set the role of point mutations was to be investigated. Terminal mismatch oligonucleotide (MR-3, 33) is shown more stable state than central mismatch oligonucleotide(MR-2, 22). And 1-mer mismatch oligonucleotide(MR-2 or 3) is shown more stable state than 2-mer mismatch oligonucleotide(MR-22 or 33).

• Journal of Korean Medical Science
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• v.33 no.52
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• pp.337.1-337.14
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• 2018

Background: Mitochondrial heteroplasmy, the co-existence of different mitochondrial polymorphisms within an individual, has various forensic and clinical implications. But there is still no guideline on the application of massively parallel sequencing (MPS) in heteroplasmy detection. We present here some critical issues that should be considered in heteroplasmy studies using MPS. Methods: Among five samples with known innate heteroplasmies, two pairs of mixture were generated for artificial heteroplasmies with target minor allele frequencies (MAFs) ranging from 50% to 1%. Each sample was amplified by two-amplicon method and sequenced by Ion Torrent system. The outcomes of two different analysis tools, Torrent Suite Variant Caller (TVC) and mtDNA-Server (mDS), were compared. Results: All the innate heteroplasmies were detected correctly by both analysis tools. Average MAFs of artificial heteroplasmies correlated well to the target values. The detection rates were almost 90% for high-level heteroplasmies, but decreased for low-level heteroplasmies. TVC generally showed lower detection rates than mDS, which seems to be due to their own computation algorithms which drop out some reference-dominant heteroplasmies. Meanwhile, mDS reported several unintended low-level heteroplasmies which were suggested as nuclear mitochondrial DNA sequences. The average coverage depth of each sample placed on the same chip showed considerable variation. The increase of coverage depth had no effect on the detection rates. Conclusion: In addition to the general accuracy of the MPS application on detecting heteroplasmy, our study indicates that the understanding of the nature of mitochondrial DNA and analysis algorithm would be crucial for appropriate interpretation of MPS results.

• Analytical Science and Technology
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• v.8 no.4
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• pp.887-894
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• 1995

Toward the development of universal, sensitive, and convenient method of DNA (or RNA) detection, two kinds of electrochemically active DNA ligands. acridine - viologen and oligonucleotide - ferrocene conjugate, were prepared. Thermodynamic and electrochemical study revealed that these probes bound strongly to DNA, and showed a typical cyclic voltammograms, indicating a potential for use as a reversible electrochemical labelling agent for DNA. Especially, using the electrochemically active oligonucleotide, we have been able to demonstrate the detection of DNA at femtomole levels by HPLC equipped with ordinary electrochemical detector (ECD). These results lead to the conclusion that the redox-active probes are very useful for the microanalysis of nucleic acid due to the stabilily of the complexes, high detection sensitivity, and wide applicability to the target structures (single- and double strands) and sequences.

• Journal of Ginseng Research
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• v.13 no.1
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• pp.42-48
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• 1989

The binding of bay region diol-epoxides of polycyclic aromatic hydrocarbons (PAHs) to target tissue DNA is thought to be essential for the initiation of cancer by these compounds. In this study we investigated the effect of polyacetylenes such as panaxynol and panaxydol on the formation of benzo(a)pyreno (BP)-metabolite-DNA adduct in the liver of ICR mice. Treatment of mice by i.p. administration of polyacetylenes produced a marked reduction in BP metabolite binding to DNA in vitro. Following i.v. administration of (3H)BP(300, ${\mu}$Ci/21 nmoles/0.1 nt DMSO) to mice, radioactivity was detected in the DNA of the liver in vivo. The result of tentative identification of the 4 peaks between the two standard markers for high pressure liquid chromatography showed that the peaks. I, II, III, and IV were BP-phenol oxide-DNA adduct (or BP-diol-epoxide-dCyt. adduct), (-) BP$.$diolepoxide I:dGuO adduct, (+) BP-diol-epoxide I: dGuo adduct, and BP-diol-epoxide II:dGuO adduct, respectively. The minor adduct, (-) BP-diol epoxide I: dGuo was reduced to 6971 of the amount of the control, while the major adduct, (+) BP-diolepoxide I: dGuO(peak II) which was produced from (-) BP-7, 8-diol was reduced to 78% of that of the control. The amount of the minor adduct, BP-diol-epoxide II:dGuo adduct(peak IV) which formed from (+) BP-7, 8-diol was 58% of the control. These results show that the panaxydol is more related to inhibition of the formation of the minor ad- ducts than of the major adducts, which were generally produced from ($\pm$) BP-7, 8-dihydro-dials.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.123-130
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• 1995

DNA topoisomerase I have been shown to be important therapeutic target in cancer chemotherapy. Chemotaxonomy and numerical identification were carried out for an isolate strain No.7489 producing an antibiotic that inhibits DNA topoisomerase I activity. The genus of strain No.7489 was determined as Streptomyces sp. from culture, morphological and chemotaxonomic data. Thirty-nine taxonomic unit characters were tested and the data were analyzed numerically using the TAXON program. The isolate was best matched to Streptomyces melanosporofaciens in the major cluster 32 of Streptomyces. Therefore, it was concluded that the isolate was identified to be a member of Streptomyces melanosporofaciens.

• Proceedings of the Korean Information Science Society Conference
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• 2005.11b
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• pp.259-261
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• 2005

분류 방법으로서의 SVM(Support Vector Machine)은 커널 방법과 함께 사용됨으로써 그 유용성을 크게 향상시켰다. 커널 방법은 일반적으로 입력 데이터의 자질(feature)로 나타내는 공간으로부터 높은 차원의 공간으로 데이터를 사상(mapping)시키는 역할을 하게 되나, 기본적으로는 데이터간에 새로운 거리(metric)를 부설해주는 역할을 하는 것이다. 지금까지 나온 다양한 커널 방법은 구조화된(structured) 데이터에 대해 커널 형태로 거리를 부여하는 방법을 제시한다. 본 논문에서는 DNA의 작용을 모델링하여 만든 새로운 커널이 miRNA(micro RNA)와 mRNA(messenger RNA)쌍에 대한 발현 여부를 분류해 내기 위해 커널 형식으로 거리를 부여하는 방법을 보인다. 이 방법은 실리콘 컴퓨터가 아닌 실제 DNA분자로 실험할 수 있도록 설계된 것을 고려할 때 여러 종류의 DNA 코드를 분석하는 데 사용될 수 있는 새로운 분자컴퓨팅 방법이다.