• Molecules and Cells
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• v.19 no.2
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• pp.246-255
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• 2005

Retroviral integrases insert viral DNA into target DNA. In this process they recognize their own DNA specifically via functional domains. In order to analyze these functional domains, we constructed six chimeric integrases by swapping domains between HIV-1 and HFV integrases, and two point mutants of HFV integrase. Chimeric integrases with the central domain of HIV-1 integrase had strand transfer and disintegration activities, in agreement with the idea that the central domain determines viral DNA specificity and has catalytic activity. On the other hand, chimeric integrases with the central domain of HFV integrase did not have any enzymatic activity apart from FFH that had weak disintegration activity, suggesting that the central domain of HFV integrase was defective catalytically or structurally. However, these inactive chimeras were efficiently complemented by the point mutants (D164A and E200A) of HFV integrase, indicating that the central domain of HFV integrase possesses potential enzymatic activity but is not able to recognize viral or target DNA without the help of its homologous N-terminal and C-terminal domains.

• Proceedings of the KIEE Conference
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• 2002.07c
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• pp.1569-1571
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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. A new electrochemical biosensor is described for voltammetric detection of gene sequence related to probe oligonucleotide of bacterium Escherichia coli O157:H7. The biosensor involves the immobilization of a 18-mer probe oligonucleotide, which is complemetary to a specific gene sequence related to Escherichia coli O157:H7 on a gold electrode through specific adsorption. The probe oligonucleotide was used to determine the amount of target oligonucleotide in solution using mitoxantrone(MTX) as the electrochemical indicators. The cathodic peak currents $(I_{peak})$ of MTX were linearly related to the concentration of the target oligonucleotide sequence in the range $1[{\mu}M]{\sim}0.1[nM]$. The detection limit of this approach was 0.01[nM]. In addition, these indicators were capable of selectivity discriminating against various mismatching condition.

• Journal of Veterinary Science
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• v.21 no.1
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• pp.4.1-4.9
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• 2020

Fast and accurate detection of viral RNA pathogens is important in apiculture. A polymerase chain reaction (PCR)-based detection method has been developed, which is simple, specific, and sensitive. In this study, we rapidly (in 1 min) synthesized cDNA from the RNA of deformed wing virus (DWV)-infected bees (Apis mellifera), and then, within 10 min, amplified the target cDNA by ultra-rapid qPCR. The PCR products were hybridized to a DNA-chip for confirmation of target gene specificity. The results of this study suggest that our method might be a useful tool for detecting DWV, as well as for the diagnosis of RNA virus-mediated diseases on-site.

• Bulletin of the Korean Chemical Society
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• v.27 no.5
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• pp.657-662
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• 2006

Identification of accessible sites in targeted RNAs is a major limitation to the effectiveness of antisense oligonucleotides. A class of antisense oligodeoxynucleotides, known as the “10-23” DNA enzyme or DNAzyme, which is a small catalytic DNA, has been shown to efficiently cleave target RNA at purine-pyrimidine junctions in vitro. We have designed a strategy to identify accessible cleavage sites in the target RNA, which is hepatitis C virus nonstructural gene 3 (HCV NS3) RNA that encodes viral helicase and protease, from a pool of random DNAzyme library. A pool of DNAzymes of 58 nucleotides-length that possess randomized annealing arms, catalytic core sequence, and fixed 5'/3'-end flanking sequences was designed and screened for their ability to cleave the target RNA. The screening procedure, which includes binding of DNAzyme pool to the target RNA under inactive condition, selection and amplification of active DNAzymes, incubation of the selected DNAzymes with the target RNA, and target site identification on sequencing gels, identified 16 potential cleavage sites in the target RNA. Corresponding DNAzymes were constructed for the selected target sites and were tested for RNA-cleavage in terms of kinetics and accessibility. These selected DNAzymes were effective in cleaving the target RNA in the presence of $Mg^{2+}$. This strategy can be applicable to identify accessible sites in any target RNA for antisense oligonucleotides-based gene inactivation methods.

• Journal of Microbiology and Biotechnology
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• v.23 no.1
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• pp.22-28
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• 2013

Xo2276 is a putative transcription activator-like effector (TALE) in Xanthomonas oryzae pv. oryzae (Xoo). Xo2276 was expressed with a TAP-tag at the C-terminus in Xoo cells to enable quantitative analysis of protein expression and secretion. Nearly all TAP-tagged Xo2276 existed in an insoluble form; addition of rice leaf extracts from a Xoosusceptible rice cultivar, Milyang23, significantly stimulated secretion of TAP-tagged Xo2276 into the medium. In a T3SS-defective Xoo mutant strain, secretion of TAPtagged Xo2276 was blocked. Xo2276 is a Xoo ortholog of Xanthomonas campestris pv. vesicatoria (Xcv) AvrBs3 and contains a conserved DNA-binding domain (DBD), which includes 19.5 tandem repeats of 34 amino acids. Xo2276- DBD was expressed in E. coli and purified. Direct in vitro recognition of Xo2276-DBD on a putative target DNA sequence was confirmed using an electrophoretic mobility shift assay. This is the first study measuring the homologous expression and secretion of Xo2276 in vitro using rice leaf extract and its direct in vitro binding to the specific target DNA sequence.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.17-17
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• 2005

In this presentation, we will discuss several recent achievements developed in my laboratory for microarray-based diagnosis of human genetic mutations including HNF-1 and BRCA1 mutations. To determine the presence of the genetic mutations in a human sample, we prepared allele-specific oligonucleotide chips from selected mutation sites and generated target probes using a tow-step method for Cy-3 DNA $samples^{1)}$ or in vitro transcription of promoter-tagged PCR products for Cy-3 RNA $samples^{2)}$. Hybridization of the target probes to the chips successfully identified all of the genotypes for the tested sites. For more reliable diagnosis, we also employed single base extension (SBE) reaction and zip-code microarray technique for our strategy. Particularly we developed an efficient PNA zip-code microarray for the detection of $HNF-1{\alpha}$ $mutations^{3)}$. Using multiplex SBE reactions and zip-code strategy, we were able to correctly diagnose several mutation sites in exon 2 of $HNF-1{\alpha}$ with a wild-type and mutant including a MODY3 patient. These works represent successful applications of DNA microarray technology for the diagnosis of human genetic mutations.

• BMB Reports
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• v.35 no.5
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• pp.532-535
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• 2002

To establish a method to evaluate the quality of the printed microarray and DNA fragments' immobilization. The target gene fragments that were made with the restriction display PCR (RD-PCR) technique were printed on a superamine modified glass slide, then immobilized with UV cross-linking and heat. This chip was hybridized with universal primers that were labeled with cy3-dUTP, as well as cDNA that was labeled with cy3-dCTP, as the conventional protocol. Most of the target gene fragments on the chip showed positive signals, but the negative control showed no signal, and vice versa. We established a method that enables an effective evaluation of the quality of the microarrays.

• Biomedical Science Letters
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• v.18 no.1
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• pp.29-34
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• 2012

Streptomycetes are gram-positive filamentous bacteria that are well-known for producing a vast array of bioactive compounds, including more than 70 % of commercially important antibiotics. For the research about Streptomyces sp., the protoplast and electroporation transformation method have been the general techniques for the construction of transformants. However, these techniques have low efficiency and are time-consuming. Another option is intergenic conjugation, which is used for DNA transfer using methylation-deficient E. coli as a DNA donor to avoid the methylated-DNA-dependent restriction systems of actinomycetes. This conjugation method has been widely improved and applied to many other actinomycetes. In this research, an effective transformation procedure for the construction of expression vector by using gateway system was established to avoid limit of restriction enzyme site for cloning of target gene based on transconjugation by Escherichia coli ET12567/pUZ8002 with a pSET152 integration vector.

• Journal of Veterinary Clinics
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• v.15 no.1
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• pp.140-145
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• 1998

The present study was carried out for the rapid and accurate detection of Anaplasma marginale in cattle using Polymerase Chain Reaction. One pair of primer, BAP-2 and AL34S, were designed to amplify a 409 Up fragment of the A marginale membrane surface protein encoding beta($msp{\beta}l$) gene with a hilly sensitive and specific PCR. A marginale isolated from naturally infected calf in Chonbuk area were used to obtain target genomic DNA for PCR. This study showed that a 409 bp of $msp{\beta}l$ gene fragment could be detected as little as 15 fg of purified A marginale genomic DNA. The amplified fragment with PCR was checked for the identification of $msp{\beta}l$ gene by enzyme restriction and sequencing. Also, the target DNA extracted directly from blood were used in the PCR reactions without prior purification to shorten the detection time. The PCR in the present study was considered convenient and rapid method for the detection of A marginale in whole blood of infected cattle.

• Korean Journal of Microbiology
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• v.39 no.3
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• pp.128-134
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• 2003

Transformation-associated recombination (TAR) cloning is based on co-penetration into yeast spheroplasts of genomic DNA along with TAR vector DNA that contains 5'- and 3'-sequences (hooks) specific for a gene of interest, followed by recombination between the vector and the human genomic DNA to establish a circular YAC. Typically, the frequency of recombinant insert capture is 0.01-1% for single-copy genes by TAR cloning. To further refine the TAR cloning technology, we determined the effect of GC content on target hooks required for gene isolation utilizing the $Tg\cdot\AC$ mouse transgene as the targeted region. For this purpose, a set of vectors containing a B1 repeated hook and Tg AC-specific hooks of variable GC content (from 18 to 45%) was constructed and checked for efficiency of transgene isolation by radial TAR cloning. Efficiency of cloning decreased approximately 2-fold when the TAR vector contained a hook with a GC content ～${\leq}23$% versus ～40%. Thus, the optimal GC content of hook sequences required for gene isolation by TAR is approximately 40%. We also analyzed how the distribution of high GC content (65%) within the hook affects gene capture, but no dramatic differences for gene capturing were observed.