• 대한화장품학회지
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• 제21권1호
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• pp.53-66
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• 1995

우유 단백질인 ${\gamma}$- 카제인 유전자는 여러 호르몬들에 의해서 임신기간과 비유기기간 동안에 동물의 유선조직에서 발현되는 카제인 유전자 집단의 하나이다. 우유 단백질 유전자의 유도를 조절하는 호르몬에 관한 메커니즘 설명으로 생쥐 ${\gamma}$- 카제인 유전자가 분석되었고 특성을 밝혔다. ${\gamma}$- 카제인 유전자는 박테리오파지 EMBL 3벡터에 삽입된 제놈 도서관으로부터 ${\gamma}$- 카제인 cDNA를 probe로 사용하여 스크린하여 하나의 클론을 얻었다. ${\gamma}$- 카제인 cDNA를 probe는 부분적으로 염기배열을 밝혔으며 ATC 개시 암호와 5'-noncoding 부위를 포함하고 있다. 클론된 제놈 DNA는 제한효소 Sal I에 의해서 ENBL 3벡터로부터 분리 되었다. 3개의 DNA밴드들을 관찰할 수 있었다. 각각의 크기는 28Kb, 14Kb 그리고 9Kb 이다. 따라서 삽입된 DNA의 크기는 대략적으로 23Kb 이다. Southern blot 분석 결과, 클론된 제놈 DNA는 cDNA 5' 발단 부위를 합성한 oilgonucleotides(40 mer)와는 결합되지 않음을 보여주고, 그러나 ${\gamma}$- 카제인 cDNA와는 결합되는 것을 보여 준다. Pormoter 부위를 포함하는 ${\gamma}$- 카제인 제놈 DNA는 cDNA 5' 말단 부위의 합성된 probe에 의해서 생쥐 제놈 도서관으로 부터 스크린하여 현재 29개의 클론을 얻었다.

• Restorative Dentistry and Endodontics
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• 제20권2호
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• pp.645-654
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• 1995

Porphyromonas endodontalis is a black-pigmented anaerobic Gram negative rod which is associated with endodontal infections. It has been isolated from infected dental root canals and submucous abscesses of endodontal origin. DNA probe is an available alternative, offering the direct detection of a specific microorganism. Nucleic-acid probes can be off different types: whole different: whole-genomic, cloned or oligonucleotide probes. Wholegenomic probes are the most sensitive because the entire genome is used for possible hybridization sites. However, as genetically similar species of bacteria are likely to be present in specimences, cross-reactions need to be considered. Cloned probes are isolated sequences of DNA that do not show cross-reactivity and are produced in quantity by cloning in a plasmid vector. Cloned probes can approach the sensitivity found with whole-genomic probes while avoiding known cross-reacting species. Porphyromonas endodontalis ATCC 35406 (serotype $O_1K_1$) was selected in this experiment to develop specific cloned DNA probes. EcoR I-digested genomic DNA fragments of P. endodontalis ATCC 35406 were cloned into pUC18 plasmid vector. From the E. coli transformed with the recombinant plasmid 4 clones were selected to be tested as specific DNA probes. Restriction-digested whole-genomic DNAs prepared from P. gingivalis 38(serotype a), W50(serotype b), A7A1-28(serotype C), P. intermedia 9336(serotype b), G8-9K-3(serotype C), P. endodontalis ATCC 35406(serotype $O_1K_1$), A. a Y4(serotype b), 75(serotype a), 67(serotype c), were each seperated on agarose gel electrophoresis, blotted on nylon membranes, and were hybridized with digoxigenin-dUTP labeled probe. The results were as follows: 1. Three clones of 1.6kb(probe e), 1.6kb(probe f), and 0.9kb(probe h) in size, were obtained. These clones were identified to be a part of the genomic DNA of P. endodontalis ATCC 35406 judging from their specific hybridization to the genomic DNA fragments of their own size on Southern blot. 2. The clones of 4.9kb(probe i) was identified to be a part of the genomic DNA of P. endodontalis ATCC 35406. but not to specific for itself. It was hybridized to P. gingivalis A7A1-28, P. intermedia G89K-3.

• KSBB Journal
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• 제18권3호
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• pp.190-196
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• 2003

임신이나 배란진단과 같이 가정에서 직접 사용할 수 있는 형태의 membrane strip 크로마토그래피 방법을 이용하여 식중독 균 DNA 분석시스템을 개발하였다. 분석물질로서는 빈번하게 발생하고 있는 식중독 미생물 중에서 S. typhimurium을 선택하였으며, Salmonella 종에 특이한 유전자 부위인 invA 유전자 분석을 목표로 하였다. 우선 이 유전자는 본 실험실 내에서 설계한 primer 쌍을 사용한 PCR 공정을 통하여 증폭되었다. 이렇게 증폭한 산물을 본 연구자들이 설계한 DNA probe와의 hybridization을 통하여 분석함으로써 전통적으로 사용하고 있는 전기영동 분석법의 단순히 분자크기에 의한 분리법과 비교하여 특이한 분석을 할 수 있었다. 이때 PCR 후 과량으로 잔존하는 primer를 별도로 제거하지 않고 hybridization을 수행할 수 있도록 특별하게 DNA probe를 설계하였다. 또한, probe가 증폭된 DNA와 hybridization 하였을 때 고체표면에 의한 간섭효과가 최소화되도록 설계 시 반영되었고 더욱이 streptavidin-비오틴의 결합을 이용하여 probe를 고정함으로써 고상에서의 상호작용이 더욱 용이하도록 배려하였다. 이러한 분석방법을 이용하여 분석한 결과 시료첨가 후 20-40분 정도에 최소 $10^3$cfu/mL (10 cells/system) 농도의 박테리아를 분석할 수 있었다. 이러한 결과는 일반적으로 사용하는 전기영동법보다 약 10배정도 더 민감한 결과일 뿐만 아니라 실험실 기기를 사용하지 않고 분석을 수행함으로써 분석시료가 제공되는 현장에서도 식중독 균의 탐지가 가능하게 되었다.

• Journal of Periodontal and Implant Science
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• 제32권2호
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• pp.269-280
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• 2002

The purpose of this study is to develop species-specific DNA probes and polymerase chain reaction (PCR) primers for detection and identification of Prevotella nigrescens (P. nigrescens) 9336. This study procedure includes (1) whole-genomic DNA extraction of P. nigrescens 9336 (2) construction of the genomic DNA library, (3) screening of strain-specific DNA probe by reverse Dot Hybridization method, (4) confirmation of strain-specific DNA probe by Southern blot analysis, (5) determination of nucleotide sequences of strain-specific DNA probe. Thirty-five restriction fragments of P. nigrescens 9336 genomic DNA digested with the Hind III were obtained. Reverse dot hybridization and Southern blot analysis data showed that three of them, Pn10, Pn23, and Pn35, could be P. nigrescens 9336-specific DNA probes. These data indicated that these DNA probes could be useful in detection and identification of the P. nigrescens 9336.

• Molecules and Cells
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• 제27권2호
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• pp.205-209
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• 2009

The loci of the 5S and 45S rRNA genes were localized on chromosomes in five species of Capsicum, namely, annuum, chacoense, frutescens, baccatum, and chinense by FISH. The 5S rDNA was localized to the distal region of one chromosome in all species observed. The number of 45S rDNA loci varied among species; one in annuum, two in chacoense and frutescens, and chinense, and four in baccatum, with the exceptions that 'CM334' of annuum had three loci and 'tabasco' of frutescens gad one locus. 'CM334'-derived BAC clones, 384B09 and 365P05, were screened with 5S rDNA as a probe, and BACs 278M03 and 262A23 were screened with 25S rDNA as a probe. Both ends of these BAC clones were sequenced. FISH with these BAC probes on pachytenes from 'CM334' plant showed one 5S rDNA locus and three 45S rDNA loci, consistent with the patterns on the somatic chromosomes. The 5S rDNA probe was also applied on extended DNA fibers to reveal that its coverage measured as long as 0.439 Mb in the pepper genome. FISH techniques applied on somatic and meiotic chromosomes and fibers have been established for chili to provide valuable information about the copy number variation of 45S rDNA and the actual physical size of the 5S rDNA in chili.

• 한국양식학회:학술대회논문집
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• 한국양식학회 2003년도 추계학술발표대회 논문요약집
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• pp.132-133
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• 2003

The marine dinoflagellate genus Alexandrium comprise PSP producing A. acatenella, A. angustitabuzatum, A. catenella, A. fundyense, A. minutum, A. ostenfezdii, A. tamiyavanichii and A. tamarense. In monitoring toxic Alexandrium, rapid and exact species identification is one of the significant prerequisite work, however we have suffered confusion of species definition in Alexandrium. To surmount this problem, we chose DNA probing, which has long been used as an alternative for conventional identification methods, primarily relying on morphological approaches using microscope in microbial field. Oligonucleotide DNA probes targeting rRNA or rDNA have been commonly used in diverse studies to detect and enumerate cells concerned as a culture-indetendent powerful tool. Despite of the massive literature on the HAB species containing Alexandrium, application of DNA probing for species identification and detection has been limited to a few documents. DNA probes of toxic A. tamarense, A. catenella and A. tamiyavanichii, and non-toxic A. affine, A. fraterculus, A. insuetum and A. pseudogonyaulax were designed from LSU rDNA D1-D2, and applied to whole cell-FISH. Each DNA probes reacted only the targeted Alexandrium cells with very high species-specificity within Alexandrium. The probes could detect each targeted cells obtained from the natural sea water samples without cross-reactivity. Labeling intensity varied in the growth stage, this showed that the contents of probe-targeted cellular rRNA decreased with reduced growth rate. Double probe TAMID2S1 achieved approximately two times higher fluorescent intensity than that with single probe TAMID2. This double probe did not cross-react with any kinds of microorganisms in the natural sea waters. Therefore we can say that in whole-cell FISH procedure this double DNA probe successfully labeled targeted A. tamiyavanichii without cross-reaction with congeners and diverse natural bio-communities.

• 한국작물학회지
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• 제38권3호
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• pp.275-282
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• 1993

본 연구는 한국 옥수수의 $\alpha$-amylase의 유전자 클로닝을 주된 목표로 하여 수행되었다. 이를 위하여 여러 식물체의 $\alpha$-amylase 염기서열로 부터 잘 보존된 부분을 참고로 oligonucleotlde probe 및 PCR primer를 설계, 합성하고, 옥수수의 유묘로부터 전체 RNA를 분리하여 northern blot analysis를 통하여 확인한 다음, 이로부터 첫 번째 가닥 cDNA를 만든 후, 여기서 얻은 RNA : DNA hybrid를 주형으로 한 polymerase chain reaction을 통하여 길이 가 약 500bp되 는 PCR 산물을 얻었다. 이를 클로닝하기 위해 pUC19을 클로닝 백터로 사용하여 재조합 플라스미드인 $\ulcorner$pZM$\alpha$'$\lrcorner$를 만들었다. 합성 probe를 이용, Southern blot analysis한 결과, $\ulcorner$pZM$\alpha$'$\lrcorner$가 옥수수 mRNA로 부터 증폭된 DNA의 일부분을 갖고 있음을 확인하였으며, 그 길이는 PCR 산물과 같은 500bp가량 되는 것으로 나타났다.

• 한국기계가공학회지
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• 제12권4호
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• pp.29-33
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• 2013

Here we report a partially aminated micropattern via direct functionalization and examine eleven different solution-phase probe DNAs hybridizing with the same target DNA on both hydrogen and oxygen terminated diamond. The hybridization intensities determined by epifluorescence microscopy were compared and are influenced strongly by the negatively charged surface and mismatched position of its sequence with immobilized probe DNA.

• Asian Pacific Journal of Cancer Prevention
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• 제15권24호
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• pp.10647-10652
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• 2015

Background: The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. Materials and Methods: We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. Results: It was found that the sensitivity of Sanger reached 0.5% with COLD-PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. Conclusions: It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

• Bulletin of the Korean Chemical Society
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• 제26권3호
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• pp.379-383
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• 2005

This research aims to develop DNA chip array without an indicator. We fabricated microelectrode array by photolithography technology. Several DNA probes were immobilized on an electrode. Then, indicator-free target DNA was hybridized by an electrical force and measured electrochemically. Cyclic-voltammograms (CVs) showed a difference between DNA probe and mismatched DNA in an anodic peak. Immobilization of probe DNA and hybridization of target DNA could be confirmed by fluorescent. This indicator-free DNA chip microarray resulted in the sequence-specific detection of the target DNA quantitatively ranging from $10^{-18}\;M\;to\;10^{-5}$ M in the buffer solution. This indicator-free DNA chip resulted in a sequence-specific detection of the target DNA.