• Title/Summary/Keyword: hybridization

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Molecular Cloning of $\beta$-Galactosidase Gene from Neisseria lactamica 2118 into Escherichia coli MC 1061 (Neisseria lactamica 2118의 $\beta$-galactosidase 유전자의 대장균으로의 클로닝)

  • Lee, Jong-Su
    • The Journal of Natural Sciences
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    • v.5 no.1
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    • pp.37-45
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    • 1992
  • The gene coding for $\beta$-galactosidase of Neisseria lactamica 2118 was cloned into Escherichia coli MC 1061. The isolated 6.5 Kb EcoR I fragement and 7.2 Kb BamH I fragment of chromosomal DNA in Southern hybridization were ligated to a vector plasmid pBR322 and then transformed into Escherichia coli MC 1061 cells. Finally, I obtained three clones as $\beta$-galactosidase positive clone by colony hybridization and Southern hybridization($\beta$-galactosidase probe: lac Z gene of pMC1871). Three recombinant plasmids(pNL.13. 17 and 24) were found to contain the 7.2Kb BamH I fragment originated from Neisseria lactamica 2118 chromosomal DNA by Southern hybridization and pNL 24 was showed high homology to probe especially and also its physical map was constructed.

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Differentiation of four Mycobacterium Species using DNA-DNA Hybridization Method using Specific Probes

  • Kweon, Tae-Dong;Bai, Sun-Joon;Hong, Seong-Karp
    • Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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    • 2013.05a
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    • pp.1012-1014
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    • 2013
  • DNA-DNA hybridization method with four oligonucleotide-specific probes was used simultaneously for differentiation and identification of four Mycobacterium species (Mycobacterium tuberculosis, M. avium, M. intracellulare, and M. kansasii). This DNA-DNA hybridization method with 4 oligonucleotide-specific probes, which targets in the rpoB region of 4 Mycobacteria species, respectively, was tested on 322 clinical isolates. Using DNA-DNA hybridization method, we detected M. tuberculosis (282 strains), M. avim (7 strains), M. intracellulare (9 strains), and M. kansasii (3 strain) from 322 clinical isolates. This result was compared with conventional biochemical test and rpoB DNA sequence analysis of this clinical isolates. We confirmed identification of Mycobacterium tuberculosis, M. avium, M. intracellulare, and M. kansasii with high sensitivity (100 %) and specificity (100 %). This DNA-DNA hybridization method could be performed within 4 hours at least. Therefore, we suggest that DNA- DNA hybridization method using 4 rpoB DNA probes of Mycobacteria could be used for accurate, rapid, convenient detection and identification of Mycobacterium tuberculosis, M. avium, M. intracellulare, and M. kansasii in clinical samples.

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Real-Time Detection of DNA Hybridization Assay by Using Evanescent Field Microscopy

  • Kim, Do-Kyun;Choi, Yong-Sung;Murakami, Yuji;Tamiya, Eiichi;Kwon, Young-Soo
    • KIEE International Transactions on Electrophysics and Applications
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    • v.11C no.3
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    • pp.85-90
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    • 2001
  • The determination of DNA hybridization reaction can apply the molecular biology research, clinic diagnostics, bioengineering, environment monitoring, food science and other application area. So, the improvement of DNA detection system is very important for the determination of this hybridization reaction. In this study, we report the characterization of the probe and target oligonucleotide hybridization reaction using the evanescent field microscopy. First, we have fabricated DNA chip microarray. The particles which were immobilized oligonucleotides were arranged by the random fluidic self-assembly on the pattern chips, using hydrophobic interaction. Second, we have detected DNA hybridization reaction using evanescent field microscopy. The 5'-biotinylated probe oligonucleotides were immobilized on the surface of DNA chip microarray and the hybridization reaction with the Rhodamine conjugated target oligonucleotide was excited fluorescence generated on the evanescent field microscopy. In the foundation of this result, we could be employed as the basis of a probe olidonucleotide, capable of detecting the target oligonucleotide and monitoring it in a large analyte concentration range and various mismatching condition.

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Comparison of Acid-Fast Staining, PCR, LCR, PCR-Hybridization for Detection of Mycobacterum Tuberculosis in Clinical Specimens (임상 검체에서 결핵균 검출을 위한 항산성염색, PCR, LCR, PCR-Hybridization 검사법 간의 비교)

  • Choi, Jong-Rak;Lim, Jong-Baeck;Kim, Hyung-Jung
    • Tuberculosis and Respiratory Diseases
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    • v.49 no.3
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    • pp.281-289
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    • 2000
  • Background : Mycobacterial culture is a confirmatory test to detect. M. tuberculosis, but it takes at least 6 weeks to diagnose. PCR is a rapid and sensitive method, but it is known that PCR has a high false positive rate due to contamination, and a high false negative rate due to inhibitors. It is also known that LCR and PCR-Hybridization, recently developed methods, are more specific methods than PCR in terms of detecting M. tuberculosis. In this study, we estimated the clinical utility of in house PCR, LCR and PCR-Hybridization for the detection of M. tuberculosis. Methods : We evaluated 75 specimens, upon which M. tuberculosis culture based testing was requested, by PCR LCR, and PCR-Hybridization and compared results. Mycobacterial culture was performed on 3% Ogawa media for 8 weeks, and an in house PCR, LCx Mycobacterium tuberculosis assay kit (Abbott Laboratories, North Chicago, III) and the AMPLICOR M. tuberculosis test kit (Roche Molecular Systems, Inc. Branchburg, NJ. USA). Results : In the view of the culture results, the sensitivities of the three tests were 40%, 80%, and 100% and their specificities were 98.6%, 94.3%, and 94.3%. Conclusion : LCR and PCR-Hybridization are rapid and sensitive methods for detecting M. tuberculosis in clinical laboratories.

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Chromosome Rearrangements Detected by Fluorescence in situ Hybridization in Human Lymphocyte Exposed to Bleomycin (Fluorescence in situ hybridization (FISH)를 이용하여 분석한 Bleomycin에 의한 사람 림프구의 염색체 재배열)

  • 손은희;정경인;정해원
    • Environmental Mutagens and Carcinogens
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    • v.17 no.1
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    • pp.12-16
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    • 1997
  • Chromosome rearrangement induced by bleomycin were identified by fluorescence in situ hybridization with probe for chromosome 4. The frequency of color junctions, translocations, dicentric and acenttic fragments increased with bleomycin dose. Different types of balanced translocation and dicentric were scored and compared. The frequency of cells exhibiting multiple aberration was higher compared to that of cells exposed to Gamma radiation suggesting that effect of bleomycin might be similar to that of high LET radiation.

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Identification of Viral RNA by Light Microscopic in situ Hybridization (광학현미경 In Situ Hybridization에 의한 Viral RNA 증명)

  • 최원기;주경웅;김석홍
    • Biomedical Science Letters
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    • v.2 no.2
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    • pp.249-255
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    • 1996
  • In this paper, a in situ hybridization(ISH) has been used to investigate the yield of viral RNA expression from each organ tissues. It is studied to establish a rapidly, specific diagnostic method detecting rabbit haemorrhagic disease virus(RHDV) RNA in 10% formalin-fixed, paraffin-em-bedded tissues of naturally RHDV-infected rabbits using oligonucleotide probe to be made by RHDV total sequences. Biotin was used as the oligonucleotide probe marker. in situ hybridization is detected the virus genome in the cells and tissue as specifically compared with others nucleic acid hybridization method. All ISH procedure of RHDV were completed to Mi-croProbe$^{TM}$ capillary action system within 1-2 hours. In this report, RHDV was distributed widely in the cytoplasm of liver cell and the cortex of kidney but lung tissue and medulla of kidney were showed to positive reaction at locally. Although not entirely free of technical limitations, nucleic acid identification holds advantages over other diagnostic tests, including exquisite sensitivity, specificity, interchangeability and speed. It is expected that, in the immediate future viral nucleic acid detection will be a prominent part of the methods used in histopathology.

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Interspecific Hybridization of the Korean Native Bumblebee Bombus hypocrita sapporoensia and the European Bumblebee B. terrestris

  • Yoon, Hyung-Joo;Park, In-Gyun;Lee, Kyeong-Yong;Kim, Mi-Ae;Jin, Byung-Rae
    • International Journal of Industrial Entomology and Biomaterials
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    • v.23 no.1
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    • pp.167-174
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    • 2011
  • The large bumblebee $Bombus$ $terrestris$, indigenous to Europe and adjacent areas and used extensively for high-value crop pollination, has been artificially introduced to several parts of the world. Here we show the occurrence of interspecific hybridization between the bumblebee species $B.$ $hypocrita$ $sapporoensia$ and $B.$ $terrestris$ under laboratory conditions. The mating and oviposition percentages resulting from the interspecific hybridization of a $B.$ $terrestris$ queen with a $B.$ $h.$ $sapporoensis$ male were higher than those resulting from the intraspecific mating of $B.$ $h.$ $sapporoensis$. Furthermore, a competitive copulation experiment indicated that the mating of $B.$ $h.$ $sapporoensis$ males with $B.$ $terrestris$ queens was 1.2-fold more frequent than the mating of these males with $B.$ $h.$ $sapporoensis$ queens. The interspecific hybridization of a $B.$ $terrestris$ queen with a $B.$ $h.$ $sapporoensis$ male produced either $B.$ $terrestris$ workers or the $B.$ $terrestris$ male phenotype, and the hybridization of a $B.$ $h.$ $sapporoensis$ queen with a $B.$ $terrestris$ male produced $B.$ $h.$ $sapporoensis$ males. Our results indicated that interspecific hybridization occurred between $B.$ $h.$ $sapporoensis$ and $B.$ $terrestris$. These results suggest that such hybridization will have a negative competitive impact and will cause genetic contamination of native bumblebees.

Detection of Chromosomal Rearrangements by Chromium in Human Lymphocyte Using Fluorescence in situ Hybridization (FISH) with Triple Combination of Composite whole Chromosome Specific Probe (FISH(fluorescence in situ hybridization)를 이용하여 분석한 크롬에 의해 유발된 염색체 이상)

  • 정해원;김수영;맹승희;이용묵;유일재
    • Environmental Mutagens and Carcinogens
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    • v.19 no.1
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    • pp.14-19
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    • 1999
  • Chromosome rearrangements induced in human lymphocyte after in vitro exposure to chromium were analysed by the use of fluorescence in situ hybridization(FISH) with triple combination of composite whole chromosome-specific probe for chromosome 1, 2 and 4. Chromosome aberrations was scored by the Protocol for Aberration Identification and Nomenclature Terminology (PAINT). Stable translocation was the most frequent type of aberrations and dicentrics and insertions were also observed. Chromium treatment enhanced the frequencies of stable translocations and color junctions in a dose-dependent manners, but no distinct increase of dicentrics and insertions was seen. The ratio of the yields of translocation to the yields of dicentric varied between 13 to 27. The presents results demonstrate fluorescent in situ hybridization (FISH) is useful for detecting chromosomal rearrangements induced by chromium.

Global Approaches to Identify Genes Involved during Infection Structure Formation in Rice Blast Fungus, Magnaporthe grisea

  • Park, Woo-Bong
    • The Plant Pathology Journal
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    • v.19 no.1
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    • pp.34-42
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    • 2003
  • The ascomycete Magnaporthe grisea is a pathogen of rice blast and is known to form specialized infection structures called appressoria for successful infection into host cells. To understand the molecular mechanism underlying infection process, appressorium-related genes were identified through global approaches including EST sequencing, differential hybridization, and sup-pression subtractive hybridization. EST database was generated on >2,000 cDNA clones randomly selected from appressorium stage cDNA library. Large number of ESTs showed homology to known proteins possibly involved in infection-related cellular development (attachment, germination, appressorium formation, and colonization) of rice blast fungus. The 1051 ESTs showing significant homology to known genes were assigned to 11 functional categories. Differential hybridization and suppression subtractive hybridization were applied to identify genes showing an appressorium stage specific expression pattern. A number of genes were selected as up-regulated during appressorium formation compared with the vegetative growing stage. Clones from various cDNA libraries constructed in different developmental stages were arrayed on slide glass for further expression profiling study. functional characterization of genes identified from these global approaches may lead to a better understand-ing of the infection process of this devastating plant disease, and the development of novel ways to protect host plant.

Preparation of Oligonucleotide Arrays with High-Density DNA Deposition and High Hybridization Efficiency

  • Park, Jeong-Won;Jung, Yong-Won;Jung, Young-Hwan;Seo, Jeong-Sun;Lee, Young-Hoon
    • Bulletin of the Korean Chemical Society
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    • v.25 no.11
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    • pp.1667-1670
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    • 2004
  • In DNA microarray produced by DNA-deposition technology, DNA-immobilization and -hybridization yields on a solid support are most important factors for its accuracy and sensitivity. We have developed a dendrimeric support using silylated aldehyde slides and polyamidoamine (PAMAM) dendrimers. An oligonucleotide array was prepared through a crosslinking between the dendrimeric support and an oligonucleotide. Both DNAimmobilization and -hybridization yields on the solid support increased by the modification with the dendrimers. The increase of the immobilization and hybridization efficiency seems to result from a threedimensional arrangement of the attached oligonucleotide. Therefore, our dendrimeric support may provide a simple and efficient solution to the preparation of DNA microarrays with high-density DNA-deposition and high hybridization efficiency.