• Title/Summary/Keyword: DNA hybridization

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DNA-DNA hybridization에 의한 Bacillus coagulans의 분류학적 연구

  • 정지관
    • Proceedings of the Korean Society for Applied Microbiology Conference
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    • 1976.10a
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    • pp.187.4-188
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    • 1976
  • 서로 다른 11주의 Bacillus coagulans와 13종의 Bacillus 속 14주를 deoxyribonucleic acid (DNA) -DNA hybridization method에 의해서 분류학적인 연구를 하였다. 사용한 B. coagulans 11주종 6주는 흙에서(일본 오사까교회) 분리했고 나머지 5주는 ATCC, IFO에서 authentic strains을 얻어서 사용했다. 사용된 B. coagulans는 Bergey's Manual(8 thed)에 의거 Grodon씨들의 방법으로 동정한 결과 B. coagulans로서 확인되었다.(중략)

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Clone Identification of Cudraria Tricuspidata and Hibiscus Syriacus by Using PCR and Southern Hybridization (PCR과 Southern hybridization을 이용한 구지뽕나무와 무궁화의 클론감별)

  • Ryu, Jang-Bal;Park, Sang-Gyu
    • Applied Biological Chemistry
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    • v.41 no.1
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    • pp.42-46
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    • 1998
  • Polymerase chain reaction (PCR) and Southern hybridization analyses were carried out to identify clones of silk worm thorn (Cudraria tricuspidata) and Rose of sharon (Hibiscus syriacus) which look like one tree with two ar three, branches or two or three different trees. For PCR five different PCR primers $(17{\sim}24\;nucleotides)$ are derived from CaMV 35S promoter, nopaline synthase terminator and coding region of thylakoid membrane protein gene. In the case of silk worm thorn, about 500 bp of PCR product was produced from DNAs of one tree or branch in the presence of 35S primer alone. Southern hybridization analysis of genomic DNAs hybridized with $^{32}P$ labeled PCR product showed that the same size of DNA fragments were hybridized with different intensities. In addition, PCR analyses using 20 different primers of OPERON 10-mer kits showed that only OPA01 primer produced PCR products of different size. These results indicate that two different trees of silk worm thorn combined to one tree. In the case of the Rose of Sharon, the same size of PCR products were produced from three different samples but Southern hybridization with the above PCR product as a probe did not show any hybridized bands. PCR analyses in the presence of OPERON 10-mers showed OPA04 and OPA13 produced different products including same sizes of products. These, results indicate that three different trees of the Rose of Sharon seem to be derived from the tree.

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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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Identification of Fusobacterium nucleatum isolated from Korean by F. nucleatum subspecies-specific DNA probes (Dot blot hybridization법을 이용한 Fusobacterium nucleatum 아종-특이 DNA 프로브의 특이성 평가)

  • Kim, Hwa-Sook;Kook, Joong-Ki
    • Journal of Korean society of Dental Hygiene
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    • v.6 no.4
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    • pp.311-324
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    • 2006
  • The purpose of this investigation was to evaluate of the specificity of Fusobacterium nucleatum subspecies-specific DNA probes using dot blot hybridization. To confirm whether the clinical isolates were F. nucleatum or not, 16S rDNA of them were cloned and sequenced. The sequencing data were used in homology search with database of GenBank. When the homology was above 98% compared with the nucleotide sequence of a certain bacteria, it was judged as the same species with the bacteria. 23 strains of F. nucleatum were isolates from subgingival plaque of periodontitis patient. The clinical isolates of F. nucleatum were classified into 10 groups using phylogenetic analysis of 16S rDNA sequence. F. nucleatum subspecies nucleatum-specific DNA probe Fu4(1.3 kb) reacted with genomic DNAs from 8 type strains of F. nucleatum and it reacted strongly with those from 8 clinical isolates. The Fp4(0.8 kb) reacted with F. nucleatum subsp. polymorphum ATCC 10953 and one clinical isolates. Fv35(1.9 kb) and Fs17(8.2 kb) probes reacted with genomic DNAs from F. nucleatum subsp. vincentii ATCC 49256 and F. nucleatum subsp. fusiform ATCC 51190, respectively. Our results showed that it is not enough to evaluate the specificity of F. nucleatum subspecies-specific DNA probes with only dot blot hybridization. Therefore, Southern blot analysis will be necessary to confirm the specificity of F. nucleatum subspecies-specific DNA probes.

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DNA Chip using Single Stranded Large Circular DNA: Low Background and Stronger Signal Intensity

  • Park, Jong-Gu
    • Biomedical Science Letters
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    • v.10 no.2
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    • pp.75-84
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    • 2004
  • Massive identification of differentially expressed patterns has been used as a tool to detect genes that are involved in disease related process. We employed circular single stranded sense molecules as probe DNA for a DNA chip. The circular single stranded DNAs derived from 1,152 unigene cDNA clones were purified in a high throughput mode from the culture supernatant of bacterial transformants containing recombinant phagemids and arrayed onto silanized slide glasses. The DNA chip was examined for its utility in detection of differential expression profile by using cDNA hybridization. Hybridization of the single stranded probe DNA were performed with Cy3- or Cy5-labeled target cDNA preparations at $60^\circ$C. Dot scanning performed with the hybridized slide showed 29 up-regulated and 6 down-regulated genes in a cancerous liver tissue when compared to those of adjacent noncancerous liver tissue. These results indicate that the circular single stranded sense molecules can be employed as probe DNA of arrays in order to obtain a precious panel of differentially expressed genes.

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Development of New DNA Chip and Genome Detection Using an Indicator-free Target DNA (비수식화 DNA를 이용한 유전자 검출 및 새로운 DNA칩의 개발)

  • Park, Yong-Sung;Park, Dae-Hee;Kwon, Young-Soo;Tomoji Kawai
    • The Transactions of the Korean Institute of Electrical Engineers C
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    • v.52 no.8
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    • pp.365-370
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    • 2003
  • This research aims to develop an indicator-free DNA chip using micro-fabrication technology. At first, we fabricated a DNA microarray by lithography technology. Several probe DNAs consisting of thiol group at their 5-end were immobilized on the gold electrodes. Then indicator-free target DNA was hybridized by an electrical force and measured electrochemically in potassium ferricyanide solution. Redox peak of cyclic-voltammogram showed a difference between target DNA and mismatched DNA in an anodic peak current. Therefore, it is able to detect various genes electrochemically after immobilization of various probe DNAs and hybridization of indicator-free DNA on the electrodes simultaneously It suggested that this DNA chip could recognize the sequence specific genes.

Molecular Cloning of Serratia rnarcescens Metalloprotease Gene into Escherichia coli (Serratia marcescens Metalloprotease 유전자의 대장균에로의 클로닝)

  • 김기석;이창원;이상열;이병룡;신용철
    • Microbiology and Biotechnology Letters
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    • v.20 no.3
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    • pp.280-288
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    • 1992
  • Molecular cloning of metalloprotease gene from Serratia marcescens ATCC 21074 into Escherichia coli JM109 was carried out. Chromosomal DNA of S. marcescens was completely digested with Hind111 and southern hybridization with a synthetic oligonucleotide probe revealed that a 50 KD metalloprotease gene was contained in 4.0 Kb chromosomal DNA fragment, 4.0 Kb chromosomal DNA fragments eluted from agarose gel were ligated with pUC19 and transformed into E. coli JM109. Nine positive clones were obtained from about $1\times 10^3$ transformants by colony hybridization. Their recombinant plasmids, pSPl and pSP2 have same chromosomal DNA fragments in pUC19 in opposite-orientations. When cloned metalloprotease gene was expressed in E. coli, about 52 KD precursor protein of metalloprotease was detected by western blot analysis from E. coli harboring a recombinant plasmid pSP2. Plasmid pSP2 showed no protease activities in E. coli but overproduced the active metalloprotease in S. rnarcescens ATCC 27117.

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Electrophoretic Karyotyping by PFGE in the Genus Fusarium (Fusarium속에서 PFGE를 이용한 Electrophoretic Karyotyping)

  • Min, Byung-Re;Jung, Jin-Sook;Choi, Yong-Keel
    • The Korean Journal of Mycology
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    • v.26 no.2 s.85
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    • pp.135-143
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    • 1998
  • Contour-clamped homogeneous electric field gel electrophoresis was used to establish electrophoretic karyotype for 10 species of Fusarium sections Sporotrichiella, Liseola, Gibbosum, Discolor and Martiella. Intact chromosomal DNA was isolated from fungal protoplast and separated under various conditions according to their size in order to improve DNA separation. The numbers of chromosome-sized DNA molecules for individual species ranged from 5-13, with individual chromosomes ranging from 0.78 Mb to 7.20 Mb in size. The total genome DNA size of each species was estimated at about 18.32 Mb to 48.20 Mb. Comparison of karyotype profiles following Southern hybridization analysis with a randomly selected genomic probe of F. oxysporum formae speciales litii was carried out.

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Ancestral Remnants in the Deoxyribonucleic Acid from Pseudomonas (Deoxyribonucleic Acid 속의 Pseudomonas로부터의 Ancestral Remnants)

  • J. De Ley;In Won Park
    • Journal of the Korean Chemical Society
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    • v.13 no.4
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    • pp.355-364
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    • 1969
  • Cross-hybridizations between DNA of two pseudomonads and a xanthomonad suggested that the three DNA types had a considerable section in common. The existence of this common part was proved by hybridization of preselected DNA, i.e. DNA resulting from a previous hybridization between any one set of two DNA types, with the third type. It was thus shown that about 50% of the DNA of the three organisms was similar. This common part was isolated in pure state and its % (G+C) was found to be indentical to the overall base composition of the native DNA. The evolutionary drift in % (G+C) could thus not be detected. The total molecular weight of the chromosornal DNA/bacterial nucleoid was determined to be 2.4 ${\times} 10^9$daltons. It can therefore be estimated that the common putida-fluorescenspelargonii DNA part consists of some 2,000 cistrons. P. putida and P. fluorescens share an additional 1,300 cistrons, and all xanthomonads share at least an additional 1,000 cistrons.

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Development of Restriction Fragment Length Polymorphism(RELP) Markers in Silkworm, Bombyx mori (누에 RFLP(제한단편 다형현상)마커 개발)

  • 고승주;김태산;이영승;황재삼;이상몽
    • Korean journal of applied entomology
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    • v.36 no.1
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    • pp.96-104
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    • 1997
  • A silkworm Bombyx mori genomic DNA library was constructed from polyphagous J111 strain and unpolyphagous $C_3$ strain to develop the genomic study by DNA makers. Genomic DNAs of two strains were digested with restriction enzyme EcoRI and ligated into pUC18. The ligated plasmids were transferred into E. coli host strain DH5$\alpha$. When the genomic DNAs were hybridized with insert DNAs from transformant, could be categorized from hybridization patterns to three groups as high repetitive sequence, moderately repetitive sequence, and low-copy number sequences. A total of 219 clones containing single or low-copy number sequence inserts were examined for any polymorphisms between two strains of J111 and $C_3$. Forty six clones showed RFLPs and 10 of these clones were used as a probe of analysis of $F_2$ population derived from crossing between J111 and $C_3$ strain. The genetic inheritance tested with each clones will be important tools to construct the genetic map of the silkworm, Bombyx mori.

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