• Title, Summary, Keyword: cDNA probe

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Detection of Japanese Encephalitis Virus by Biotinylated cDNA Probe (Biotin으로 표지된 cDNA Probe를 이용한 일본 뇌염 바이러스의 검색)

  • 황동연;신영오;임정빈
    • Korean Journal of Microbiology
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    • v.26 no.3
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    • pp.149-154
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    • 1988
  • Japanese Encephalitis Virus(JEV) can be detected conveniently by the use of biotinylated cDNA probe. To prepare biotinylated probe aminoallyl dUTP was first synthesized chemically to reverse transcribe the virial RNA. The allylamine-labeled cDNA was then converted to the biotin-cDNA by the reaction with an activated biotin ester, NHS-ACA-biotin. The JEV genomic RNA was hybridized to the biotinylated cDNA probe on nitrocellulose filter and visualized colorimetrically by streptavidin complexes with alkaline phosphatase polymer. Sensitivity of the detection system was determined by estimating the amount of the JEV genomic RNA through comparison with signals generated from the biotinylated and $^{32/P}$ -labeled probes. It was found that the biotin probe was as sensitive as $^{32/P}$ -cDNA probe which can detect 50pgs of the target RNA.

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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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Identification of the Differentially Expressed Genes of Hanwoo During the Growth Stage by Subtractive cDNA Hybridization (Subtraction 기법을 이용한 한우 성장 단계 특이 발현 유전자 탐색)

  • Jang, Y.S.;Kim, T.H.;Yoon, D.H.;Park, E.W.;Cheong, I.C.;Jo, J.K.
    • Journal of Animal Science and Technology
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    • v.44 no.1
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    • pp.13-22
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    • 2002
  • To identify the differentially expressed genes at growth stage of Hanwoo, we constructed the subtractive cDNA library from loin mRNA of 12- and 24-month old Hanwoo by PCR-based subtraction. The fourteen genes were confirmed by sequencing and reverse northern blot analysis, and they were selected as candidate of putative genes differentially expressed at the growth stage of Hanwoo. Three subtracted cDNA fragments that expressed specific signal to cDNA probe for 6-month-old loin of Hanwoo were highly homologous to those of the genes encoding EPV 20, Ca2+ATPase, and TCTP, respectively. The nine cDNA clones showed intense signal to cDNA probe from 12-month-old loin of Hanwoo, and highly homologus to those of genes encoding VCP, HSP 70, aldolase A, MSSK1, GM-2 activator protein, ryanodine receptor, acidic ribosomal phosphoprotein p1, ADP/ATP translocase, and UCP 2, respectively. Two subtracted cDNA clones that expressed specific signal to cDNA probes for 12- and 24-month-old loin of Hanwoo were detected. One of them was highly homologus to the gene encoding ferrochelatase and the other was highly homologus to the gene encoding ADRP.

Oligonucleotide Probe Selection using Evolutionary Computation in Large Target Genes (다수의 목표 유전자에서 진화연산을 이용한 Oligonucleotide Probe 선택)

  • Shin, Ki-Roo;Kim, Sun;Zhang, Byung-Tak
    • Proceedings of the Korean Information Science Society Conference
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    • pp.455-457
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    • 2003
  • DNA microarray는 분자생물학에서 널리 사용되고 있는 실험 도구로써 크게 cDNA와 oligonucleotide microarray로 나뉘어진다. DNA microarray는 일련의 DNA 서열로 이루어진 probe들의 집합으로 구성되며 알려지지 않은 서열과의 hybridization 과정을 통해 특정 서열을 인식할 수 있게 된다. O1igonucieotide microarray는 cDNA 방법과는 다르게 probe를 구성하는 서열을 제작자가 임의로 구성할 수 있기 때문에 목표 서열이 가지는 고유한 부분만을 probe 서열로 사용함으로써 비용절감과 실험의 정확도를 높일 수 있다는 장점이 있다. 그러나 현재 목표 유전자 서열에 대해 probe 집합을 생성하는 결정적인 방법은 존재하지 않으며, 따라서 넓은 해 공간에서 효과적으로 최적 해를 찾아 주는 진화 연산이 probe 선택을 위한 좋은 대안으로 사용될 수 있다[1.2]. 그러나 진화연산을 이용한 probe 선택방법에 있어서 인식하고자 하는 목표 서열의 개수가 많아질 경우, 해 공간의 크기가 커짐으로 인해 문제점이 발생할 수 있다. 따라서 본 논문에서는 다수의 목표 유전자 서열을 대상으로 한 probe 선택 방법에 일어서 보다 효율적인 진화연산 접근 방법을 소개한다. 제시된 방법은 인식하고자 하는 목표 서얼의 일부를 선택해 이를 probe 집합의 후보로 사용하며. 유전 연산자를 이용한 진화과정을 통해 최적에 가까운 probe 집합을 찾는다. 본 논문은 GenBank로부터 유전자 서열을 대상으로 제안된 방법을 실험하였으며, 축소된 목표 서열만을 이용해 probe 집합을 선택하더라도 적합한 probe 집합을 찾을 수 있었다.

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Molecular cloning of casein gane which is expressed in mammary glands (유선조직에서 특이적으로 발현되는 카제인 유전자의 클로닝(I))

  • Choe, In-Ho;Bae, Bong-Jin;Lee, Chang-Su
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.21 no.1
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    • pp.53-66
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    • 1995
  • The gene for ${\gamma}$-casein, a milk protein, is a member of a family of casein gene which is expressed in mammary glands of the animal during the late gestation and lactation periods binder the influence of various hormones. In order to elucidate tile mechanisms b)'which hormones regulate the coordinate induction of milk protein genes, the mouse ${\gamma}$-casein gene was isolated and characterized. The ${\gamma}$-casein gene was screened from a mouse genomic library constructed in bacteriophage EMBL3 with the ${\gamma}$-casein CDNA used as probe and one clone was obtained. The ${\gamma}$-casein CDNA as probe was partially sequenced and contained ATG start codon and 5'-noncoding region. The cloned genomic DNA was digested with Sal I restriction enzyme, by which the insert DNA can be isolated from EMBL3 vector. Three DNA bands were observed and the size of DNAs was approximately 28kb, 14kb and 9Kb, respectively Accordingly the size of the insert DNA was calculated with approximately 23Kb. The result of Southern blot analysis, however, showed that the cloned genomic DNA was not hybridized with the synthetic oligonucleotides (40 mer) of cDNA 5'-end region, but it was hybridized with the y -casein CDNA. This means that tile cloned y -casein gene may not contain its promoter region. The ${\gamma}$ -casein genomic DNA containing the promoter region has been screening from mouse genomic library with oligonucleotides of CDNA 5'-end region as probe, and twenty-nine clones was obtained.

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Molecular probe for identification of cysts of resting cyst of PSP-producer Alexandrium tamarense (Dinophyceae) (분자생물학적 방법을 이용하여 마비성 패류 독소를 생산하는 알렉산드륨 타마렌스 시스트 탐색)

  • Cho, Eun-Seob
    • Journal of Life Science
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    • v.13 no.2
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    • pp.163-167
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    • 2003
  • Identification of species within the toxin-producing genus Alexandrium is vital for biotoxin monitoring and mitigation decisions regarding shellfish industry. In particular, the discrimination of resting cysts of only A. tamarense from that of Alexandrium spp. is considerable important to fundamentally monitor and predict this species before vegetative cells occur in the nature. Fluorescent cTAM-F1 DNA probe was responsible to not only binding the activity of the vegetative cells in A. tamarense, but also to the resting cysts, which was treated with methanol after fixation and stained by primuline on the surface The location of fluorescence in cultured vegetative cells and resting cysts was almost at tile bottom of the nucleus. The optimal incubation temperature and time using in situ hybridization were 50-$54^{\circ}C$ and 40-60 min, respectively, to penetrate the DNA probe into cell.

Assembly of a Functional cDNA for Human Liver Growth Hormone Receptor: Cloning of Assembled hGHR cDNA (Human Liver로부터 Cloning한 cDNA성장호르몬 수용체의 기능성 검토)

  • 장규태;지선병홍;손동수;서원진삼;고교적웅
    • Journal of Embryo Transfer
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    • v.13 no.2
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    • pp.159-172
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    • 1998
  • 사람 성장호르몬 수용체(hGHR) cDNA는 PCR방법에 의하여 fagment로서 보고되어진 바 있으나, liver cDNA로 부터 전장을 cloning한 보고는 없는 실정으로 본 연구에서는 기능을 가진 약 4.6kbp의 cDNA hGHR을 cloning 하는데 성공하였다. 먼저 cloning하기 위하여 human liver mRNA와 human breast cancer tissue로부터 회수한 mRNA를 RT-PCR방법에 의하여 human cDNA library와 cloning에 필요한 probe를 제작하였다. human library mRNA는 GT-PCR방법에 의하여 증폭하여 증폭되어진 산물은 λZAP Vector를 이용하여 cDNA library를 구축하였고,screeing을 위하여 임 보고 되어진 hGHR fragment native sequence를 기초로 N-terminal부분의 primer를 설계하여 950bp의 probe를 얻는데 성공하였다. 이 probe를 이용하여 준비된 human liver cDNA library로부터 2.5$\times$10 6개의 plaque로부터 6개의 positive clone을 획득하였고, 이들중 poly Asignal인 "AATAAA"를 포함하고 있는 가장 긴 약 3.8kbp의 clone을 sequencing한 결과 open reading frame을 포함하고 있었으나, 5'부분의 결손되어 있었다. 그리하여 이 부분은 human breast cancer tissue로 부터 회수한 mRNA를 RT-PCR에 의하여 증폭하였고, sequencing결과 이미 보고되어진 native hGHR와 비교한 결과 하나의 nucleotide가 silent mutation으로 판명되었다.한편 human liver cDNA library로부터 cloning한 3.8cp의 positive clone의 5'end의 결손된 부분에 silent mutation된 PCR 산물을 연결함으로써 native hGHR와 유사한 cDNA hGHR subcloning에 성공하였다. 이러한 cDNA hGHR의 clone이 function을 가지고 있는지를 검토하기 위하여 eukaryotic 발현 vector인 pCXN2에 의거 ligation한 후 chinese hamster ovary cell[CHO-KI]에 transfect를 실시하였다. Dexamethasone은 첨가하지 않고 hGH만의 존재하에서 이들 cell을 배양시키고 cell menbrane에서 발현 여부를 판정키 위하여 hGHR monocloual antibody를 사용하여 flow cytometery해석을 실시하는 한편 125I-hGH binding assay에 의하여 hGH binding activity를 측정하였다. 최종적으로 GH signal transduction의 target genedf으로 알려져 있는 serine protease inhibitor 2.1(Spi 2.1) gene의 promotor activity를 검토한 결과 hGHR을 transfect한 CHO Cell에 있어서 hGH의 농도에 의존적으로 증가되었다. 따라서 본 실험에서 cloning한 cDNA hGHR는 native hGHR와 같은 기능을 가지는 것으로 판명되었다.것으로 판명되었다.

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Cloning of $\alpha$-Amylase Gene from Zea mays (옥수수 $\alpha$-amylase 유전자의 클로닝)

  • 김용욱;강신혜
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.38 no.3
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    • pp.275-282
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    • 1993
  • The objective of this study was to clone a partial fragment of $\alpha$-amylase from Korean maize. We designed and synthesized an oligonucleotide probe and two kinds of PCR primers based on cDNA conserved region of $\alpha$-amylase sequences from other plants. Total RNA from 3-day-old maize seedling was used as template for 1st strand cDNA synthesis and RNA-DNA hybrid was used as template for polymerase chain reaction(PCR). The product of PCR was about 0.5 kb long and inserted into pUC19. We named this recombinant plasmid as pZM$\alpha$'. The cloned fragment was certified by Southern blot analysis using labeled synthetic oligonucleotide as probe.

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Indicator-free DNA Chip Array Using an Electrochemical System

  • Park, Yong-Sung;Kwon, Young-Soo;Park, Dae-Hee
    • KIEE International Transactions on Electrophysics and Applications
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    • v.4C no.4
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    • pp.133-136
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    • 2004
  • This research aims to develop a DNA chip array without an indicator. We fabricated a microelectrode array through photolithography technology. Several DNA probes were immobilized on an electrode. Then, target DNA was hybridized and measured electrochemically. Cyclic-voltammograms (CVs) showed a difference between the DNA probe and mismatched DNA in an anodic peak. This indicator-free DNA chip resulted in a sequence-specific detection of the target DNA.

Sequence analysis and cDNA probe hybridization of the nucleocapsid(N) protein gene of transmissible gastroenteritis virus(TGEV) and porcine epidemic diarrhea virus(PEDV) (Transmissible gastroenteritis virus(TGEV)와 porcine epidemic diarrhea virus(PEDV)의 nucleocapsid(N) 단백질 유전자에 대한 염기서열 분석과 cDNA probe hybridization)

  • Park, Ji-yong;Kim, Chul-joong;Shin, Kwang-soon;Kim, Won-yong;Kang, Shien-young;Park, Yong-ho;Han, Hae-jung;Park, Yong-ha
    • Korean Journal of Veterinary Research
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    • v.35 no.3
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    • pp.515-530
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    • 1995
  • Coronaviridae에 속하는 transmissible gastroenteritis virus(TGEV)와 porcine epidemic diarrhea virus(PEDV)를 specific하게 detection할 수 있는 방법을 개발하고자 본 연구를 수행하였다. 두 바이러스 모두 RNA 바이러스이기 때문에 reverse transcription-polymerase chain reaction(RT-PCR)으로 nucleocapsid(N) protein gene의 cDNA를 증폭시켰다. SmaI으로 처리한 pTZ19R에 ligation시킨 후 염기서열을 밝히고자 sequencing하였다. 각각의 prototype virus와 비교하여 상동성을 밝혔다. 두 바이러스에 대한 cDNA probe를 제작하여 Southern blot hybridization을 실시하였다. TGEV의 경우 백신주인 P45와 병독주인 Miller strain을 사용하였다. cDNA를 증폭시키기 위해 N1/N1R과 N2/N2R 두 가지 primer를 이용한 결과, N1/N1R primer의 경우 586bp 크기의 PCR product를 얻을 수 있었고, N2/N2R primers로 582bp의 cDNA를 증폭시킬 수 있었다. PEDV 실험을 위하여 PED 임상 증상을 나타내는 분변을 이용하여 RT-PCR을 실시하였다. P2/P2R primer로 753bp의 PCR product를 얻을 수 있었다. TGEV의 두 가지 strain의 N protein gene을 sequencing하여 prototype인 Purdue strain과 염기서열 상동성을 조사한 결과, 97%이상의 높은 homology를 나타내었다. PED-V 역시 N protein gene을 sequencing하여 CV777과 염기서열 상동성을 조사한 결과 97%이상의 homology로 PEDV임을 알 수 있었다. TGEV와 PEDV의 염기서열을 비교한 결과 29%의 낮은 homology를 관찰할 수 있었다. 두 가지 바이러스의 N protein gene에 대한 cDNA probe를 제작하여 Southern blot hybridization을 한 결과, 각 바이러스에 매우 특이적 반응을 나타내었다.

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