• Proceedings of the KIEE Conference
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• 2006.07c
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• pp.1319-1321
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• 2006

This research aims to develop the multiple channel electrochemical DNA chip that has the above characteristic and be able to solve the problems. At first, we fabricated a high integration type DNA chip array by lithography technology. It is able to detect a plural genes electrochemically after immobilization of a plural probe DNA and hybridization of non-labeling target DNA on the electrodes simultaneously. It suggested that this DNA chip could recognize the sequence specific genes. It suggested that multichannel electrochemical DNA microarray is useful to develop a portable device for clinical gene diagnostic system.

• Korean Journal Plant Pathology
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• v.14 no.2
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• pp.177-183
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• 1998

A DNA fragment which is involved in tolassin production was cloned to obtain a molecular marker of Pseudomonas tolaasii, a casual agent of bacterial brown blotch disease of oyster mushroom (Pleurotus ostreatus). Tolaasin is a lipodepsipeptide toxin and known as a primary disease determinant of the P. tolaasii. It is responsible for formation of white line in agar when P. tolaasii were cultured against white line reacting organisms (WLROs). White line negative mutants (WL-) were generated by conjugation between rifampicin resistant strain of P. tolaasii and E. coli carrying suicidal plasmid pSUP2021 : : Tn5. The ability of tolaasin production of the WL- mutants was examined by hemolysis test, pathogenicity test, and high pressure liquid chromatography (HPLC) analysis of culture filtrate. All of the WL- mutants were lost the ability of tolaasin production (Tol-). Genomic library of the Tol- mutant was constructed in pLAFR3 and the cosmid clone containing Tn5 was selected. DNA fragment fro franking region of Tn5 was cloned from the plasmid and used as a probe in Southern blot. DNA-DNA hybridization with the probe to total DNA from group of bacteria ecologically similar to P. tolaasii including WLORs, fluorescent Pseudomonads isolated from oyster mushroom, P. agarici, P. gingeri, and some of other species of Psedomonas showed that some of the tested bacteria do not have any hybridized band and others have bands sowing RFLP. The cloned DNA fragment or its nucleotide sequence will be useful in detection and identification of the P. tolaasii.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.130-130
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• 2003

Gummy stem blight pathogen is very difficult not only to monitor the inoculum levels prior to host infection, and also it is destructive and hard to control in field condition. We have applied RAPD technique to elucidate the genetic diversity of the genomic DNA of Didymella bryoniae and also to generate specific diagnostic DNA probe useful for identification and detection. The 40 primers produced clear bands consistently from the genomic DNA of twenty isolates of Didymella bryoniae, and two hundred seventy-three amplified fragments were produced with 40 primers. The combined data from 273 bands was analyzed by a cluster analysis using UPGMA method with an arithmetic average program of NTSYS-PC (Version 1.80) to generate a dendrogram. At the distance level of 0.7, two major RAPD groups were differentiated among 20 strains. RAPD group (RG) I included 8 isolates from watermelon except one isolate from melon. RAPD group (RG) IV included 12 isolates from squash, cucumber, watermelon and melon.. In amplification experiment with SCAR specific primer RG1F-RG1R resulted in a single band of 650bp fragment only for 8 isolates out of 20 isolates that should be designated as RAPD Group 1. However, same set of experiment done with RGIIF-RGIIR did not result in any amplified product.. Our attempts to detect intraspecific diversity of ITS region of rDNA by amplifying ITS region and 17s rDNA region for 20 isolates and restriction digestion of amplified fragment with 12 enzymes did not reveal polymorphic band. In order to develop RAPD markers for RGIV specific primer, a candidate PCR fragment( ≒1.4kb) was purified and Southern hybridized to the amplified fragment RGIV isolates. This promising candidate probe recognized only RGIV isolates

• Korean Journal of Veterinary Research
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• v.45 no.3
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• pp.351-357
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• 2005

Here I describe a multiplex allele specific PCR-based approach for the rapid detection between Hanwoo and Holstein meat associated with Melanocortin 1 receptor (MC1R) gene. Specific and universal oligonucleotide primers were used in combination to detect the presence of a single nucleotide polymorphism within the bovine MC1R DNA sequence. The presence of the bovine MC1R gene is indicated by the production of a single control PCR product, whilst positive samples generate an alternative smaller specific product over the same region. The mutations in MC1R104 codon revealed depending on the presence or absence of an indicative fragment amplified from the wild-type allele of this codon. As little as 0.39 ng and 1.56 ng of genomic DNA of Hanwoo and Holstein could be detected by MAS-PCR assay, respectively. This technique, which is widely used in human genetic screening, provides a reliable and sensitive result that has not been documented for the identification of bovine coat color. The MAS-PCR assay approach was proven to be useful in complementing routine beef DNA analysis for differentiation of these MC1R variants and it would facilitate the screening of deceiving sales of Holstein meat in the butcher shop.

• Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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• 2006.06a
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• pp.372-373
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• 2006

In this paper, a DNA chip with a microelectrode array was fabricated using microfabrication technology. Several probe DNAs consisting of mercaptohexyl moiety at their 5 end were immobilized on the gold electrodes by DNA arrayer. Then target DNAs were hybridized and reacted with Hoechst 33258, which is a DNA minor groove binder and electrochemically active dye. Linear sweep voltammetry or cyclic voltammetry showed a difference between target DNA and control DNA in the anodic peak current values. It was derived from Hoechst 33258 concentrated at the electrode surface through association with formed hybrid. It suggested that this DNA chip could recognize the sequence specific genes.

• Research in Plant Disease
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• v.18 no.2
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• pp.73-79
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• 2012

Phytophthora katsurae is a fungal pathogen responsible for chestnut ink disease. We designed two duplex primer sets (SOPC 1F/1R+KatI 3F/5R, SOPC 1-1F/1-1R+KatI 3F/5R) to detect P. katsurae. SOPC 1F/1R and SOPC 1-1F/1-1R primer pairs were designed for sequence characteristic amplification regions (SCAR) marker, and KatI 3F/5R primer pair was used for P. katsurae-specific primer designed from internal transcribed spacer (ITS) region. To assess the sensitivity of duplex PCR, genomic DNA was serially diluted 10-fold to make the final concentrations from 1 mg/ml to 1 ng/ml. The sensitivity for two primer sets were 1 ${\mu}g/ml$ and 100 ng/ml, respectively. To find detection limits for zoospores of P. katsurae, each zoospore suspension was serially diluted 10-fold to make the final concentrations from $1{\times}10^6$ to $1{\times}10^2$ cells/ml, and then DNA was extracted. The limits of detection for all of two primer sets were $1{\times}10^5$ cells/ml. All of two primer sets were specific to P. katsurae in PCR detection and did not produce any P. katsurae-specific PCR amplicons from other 16 Phytophthora species used as the control. This study shows that duplex PCR using two primer sets might be a useful tool for rapid and efficient detection of P. katsurae.

• Parasites, Hosts and Diseases
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• v.51 no.2
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• pp.237-241
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• 2013

Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from $10^{-1}$ to $10^{-5}ng/{\mu}l$ for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of $63^{\circ}C$ by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha ($EF1{\alpha}$) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.4
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• pp.263-267
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• 2003

Magnetic nanoparticles prepared from an alkaline solution of divalent and trivalent iron ions could covalently bind protein via the activation of Nethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC). Trypsin and avidin were taken as the model proteins for the formation of protein-nanoparticle conjugates. The immobilized yield of protein increased with molar ratio of EDC/nanoparticie. Higher concentrations of added protein could yield higher immobilized protein densities on the particles. In contrast to EDC, the yields of protein immobilization via the a ctivation of cyanamide were relatively lower. Nanoparticles bound with avidin could attach a single-stranded DNA through the avidin-biotin interaction and hybridize with a DNA probe. The DNA hybridization was confirmed by fluorescence microscopy observations. Immobilized DNA on nanoparticles by this technique may have widespread applicability to the detection of specific nucleic acid sequence and targeting of DNA to particular cells.

• Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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• 2006.05a
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• pp.65-70
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• 2006

In this study, a DNA chip with a microelectrode array was fabricated using microfabrication technology. Several probe DNAs consisting of mercaptohexyl moiety at their 5 end were immobilized on the gold electrodes by DNA arrayer. Then target DNAs were hybridized and reacted with Hoechst 33258, which is a DNA minor groove binder and electrochemically active dye. Linear sweep voltammetry or cyclic voltammetry showed a difference between target DNA and control DNA in the anodic peak current values. It was derived from Hoechst 33258 concentrated at the electrode surface through association with formed hybrid. It suggested that this DNA chip could recognize the sequence specific genes.

• Journal of Microbiology and Biotechnology
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• v.12 no.6
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• pp.989-992
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• 2002

Sweet persimmon (Diospyros kaki Thunb.) is widely cultivated in the southern part of Korea and its cultivation is increasing. However, anthracnose disease caused by Colletotricuhum species is one of the major hinderances to the cultivation and production of sweet persimmon. Therefore, in the current study, PCR was used to specifically detect Colletotrichum spp., based on the sequences of the ITS II regions in the rDNA. Using the sequence data, CO-1 was designated to detect Colletotrichum together the with ITS 4 primer. The result showed that a single segment of ca. 500 bp was observed only in Colletotrichum, but not in any other fungal and bacterial isolates. The annealing temperatures and template DNA quantites were also investigated to identify optimal conditions for detection. Using these species-specific primers, a unique band was obtained at annealing temperatures ranging from $55^{\circ}C\;and\;61^{\circ}C$ and template DNA levels from 10 pg- $10{\mu}g$.