• 한국미생물·생명공학회지
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• 제14권2호
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• pp.133-137
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• 1986

원생동물인 Tetrahymena thermophila의 17S-rDNA구조 및 hygromycin 내성 기구에 대한 연구의 일부로서 hygromycin 내성변이주 hmr3의 17S-rDNA를 대장균의 vector pBR 322에 cloning하였다. 우선 rDNA는 hot phenol-cresol 용액으로 추출하여 제한효소 Hind III 처리로서 약 2.2kbp의 17S-rDNA를 agarose 전기영동상에서 분리하였다. 이를 pBR 322에 cloning하여 wild type의 17S-rDNA probe와 colony hybridization시켜 선별하였다. 그중 5-19 균주의 recombinant plasmid로부터 17S-rDNA 의 전사 orientation위치가 pBR322의 tetracyline내성 유전자 쪽으로 삽입되어 있는 것을 확인하였다.

• The Plant Pathology Journal
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• 제37권3호
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• pp.291-298
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• 2021

False smut caused by Ustilaginoidea virens is an important rice fungal disease that significantly decreases its production. In the recent past, conventional methods have been developed for its detection that is time-consuming and need high-cost equipments. The research and development in nanotechnology have made it possible to assemble efficient recognition interfaces in biosensors. In this study, we present a simple, sensitive, and selective oxidized graphene-based geno-biosensor for the detection of rice false smut. The biosensor has been developed using a probe DNA as a biological recognition element on paper electrodes, and oxidized graphene to enhance the limit of detection and sensitivity of the sensor. Probe single-stranded DNA (ssDNA) and target ssDNA hybridization on the interface surface has been quantitatively measured with the electrochemical analysis tools namely, cyclic voltammetry, and linear sweep voltammetry. To confirm the selectivity of the device, probe hybridization with non-complementary ssDNA target has been studied. In our study, the developed sensor was able to detect up to 10 fM of target ssDNA. The paper electrodes were employed to produce an effective and cost-effective platform for the immobilization of the DNA and can be extended to design low-cost biosensors for the detection of the other plant pathogens.

• KIEE International Transactions on Electrophysics and Applications
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• 제4C권4호
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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.

• 한국미생물·생명공학회지
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• 제45권4호
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• pp.358-364
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• 2017

Asymmetric PCR preferentially amplifies one DNA strand for use in DNA hybridization studies. Linear-After-The-Exponential-PCR (LATE-PCR) is an advanced asymmetric PCR method which uses innovatively designed primers at different concentrations. This study aimed to optimise LATE-PCR parameters to produce single-stranded DNA of Candida spp. and Aspergillus spp. for detection via probe hybridisation. The internal transcribed spacer (ITS) region was used to design limiting primer and excess primer for LATE-PCR. Primer annealing and melting temperature, difference of melting temperature between limiting and excess primer and concentration of primers were optimized. In order to confirm the presence of single-stranded DNA, the LATE-PCR product was hybridised with digoxigenin labeled complementary oligonucleotide probe specific for each fungal genus and detected using anti-digoxigenin antibody by dot blotting. Important parameters that determine the production of single-stranded DNA in a LATE-PCR reaction are difference of melting temperature between the limiting and excess primer of at least $5^{\circ}C$ and primer concentration ratio of excess primer to limiting primer at 20:1. LATE-PCR products of Candida albicans, Candida parapsilosis, Candida tropicalis and Aspergillus terreus at up to 1:100 dilution and after 1 h hybridization time, successfully hybridised to respective oligonucleotide probes with no cross reactivity observed between each fungal genus probe and non-target products. For Aspergillus fumigatus, LATE-PCR products were detected at 1:10 dilution and after overnight hybridisation. These results indicate high detection sensitivity for single-stranded DNA produced by LATE-PCR. In conclusion, this advancement of PCR may be utilised to detect fungal pathogens which can aid the diagnosis of invasive fungal disease.

• 한국균학회지
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• 제25권4호통권83호
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• pp.362-368
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• 1997

재조합 DNA probe를 이용하여 국내 Fusarium oxysporum 분화형간의 분류 가능성을 탐색하고 그들의 유전적 변이를 분석하였다. F. oxysporum f. sp. lycopersici, cucumerinum, fragariae, garlic, sesami 등 5종의 분화형을 공시하여 RFLP 분석을 실시하였다. Repetitive copy clone인 세종의 재조합 클론 pFC46, pFC52, pFC57을 이용하여 HindIII로 처리한 F. oxysporum의 genomic DNA에 대해 southern hybridization한 결과 나타난 밴드의 양상은 분화형에 따라 차이가 명확히 밝혀져 이들을 이용한 분류가 가능하였다. 또한 RFLP 분석 결과 f. sp. sesami는 다른 분화형에 비해 다소 변이가 심했으나 다른 분화형들은 변이가 거의 없어 f. sp. sesami를 제외한 f. sp. lycopersici 등 4종의 Fusarium oxysporum 분화형의 균주들은 채집 지역에 관계없이 대체로 유전적으로 안정되어 있는 것으로 판단되었다. Hybridization밴드의 양상에 기초하여 유전적 유연관계를 집괴 분석한 결과 각 분화형별로 유사도가 매우 높게 별도의 유사군을 형성하였다.

• 대한의생명과학회지
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• 제7권1호
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• pp.1-5
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• 2001

A cDNA of coagulation Factor IX gene has been screened from the $\lambda$gt11 human fetal liver cDNA library, and used to construct a 2.8-kb full length cDNA after recombining with the N-terminal fragment from pTZ-FIX. Human genomic DNA was isolated, digested with the restriction endonucleases, TaqI, EcoRI, and HindIII, and Southern hybridization was performed using the full length factor IX cDNA as a probe. The hybridized bands generated by the restriction endonucleases were the followings: TaqI, 0.3, 1.0, 1.6, 1.8, 2.7, 3.7, and 5.3 kb bands; EcoRI, 1.8, 4.8, 4.9, 5.5, 6.8, and 12.6 kb bands; HindIII, 4.1, 4.4, 5.2, 5.8, 7.6, and 12.5 kb bands. When the Southern bands were physically mapped along the genome, about 50-kb continuous region harboring almost all of the genomic region of Factor Ⅸ gene was covered. These results suggest a possibility of using an exonal cDNA probe to diagnose abnormalities including large deletions, insertions, and rearrangements along the genome, if there is any.

• KSBB Journal
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• 제23권5호
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• pp.460-462
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• 2008

개화시기의 수술로부터 수집한 백합화분을 aluminum oxide 미세입자와 섞어 교반에 의해 상해를 발생시켰다. 이어서 이들 화분은 signal peptide-fused epidermal growth factor (EGF) DNA를 지니는 Agrobacterium 세포로 vacuum infiltration을 시켰으며 24 hr 화분신장을 통한 배양을 실시하였다. 이들 신장 화분에서의 EGF mRNA 및 단백질 발현은 성공적으로 확인되었으며 이는 cDNA blot hybridization 및 immuno-blotting의 분석결과이다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.86-86
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• 2002

To examine the feasibility of using a sperm vector system for gene transfer, we have investigated the binding and the uptaking of foreign DNA into the sperm nucleus by PCR, in situ hybridization and LSC. We have also examined the transportation of exogenous DNA into oocytes by immunofluorescene via PCR. Sperm cells were incubated with DNA/liposome complexes (1:4 ratio) in fertilization medium with BSA or without BSA. (omitted)

• 한국식물병리학회지
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• 제14권6호
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• pp.589-593
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• 1998

DNA amplification by polymerase chain reaction (PCR) was used to specifically detect Plasmodiophora brassicae, causing clubroot of crucifers. On the basis of DNA sequence informations, an oligonucleotide primer set specific for the pathogen was designed form small subunit gene (18S-like) and internal transcribed spacer (ITS) region of ribosomal DNA. Primer ITS 5/PB-C produced an amplification product of approximately 520 bp in length with DNA from P. brassicae. However, no amplification product was produced with DNAs from several soil-borne fungi, Didymella bryoniae and Rhizopus stolonifer. Using these primers, the clubroot pathogen was readily detected from infected roots of crucifers, but not from healthy roots. Southern hybridization analysis further confirmed that the amplification product was originated from P. brassicae.

• Food Science and Biotechnology
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• 제14권3호
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• pp.416-419
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• 2005

Unknown bacterium isolated from garlic was characterized using phenotypic methods, phylogenetic analysis, DNA-DNA hybridization, and cultural methods. The strain was identified as typical leuconostoc; Gram-positive, non-sporeforming, heterofermentative, catalase-negative and spherical. Although its 16S rRNA gene sequence showed high homology to Leuconostoc argentinum DSM $8581^T$(99.8%), DNA-DNA hybridization experiments indicated it represents novel genomic species in the genus Leuconostoc. The garlic-specific leuconostoc was more resistant to antimicrobial activity of garlic compared to other common laboratory lactic acid bacteria, and was even stimulated by low concentrations (1-2%) of garlic extract supplemented in trypticase soy broth. Growth stimulation was concentration-dependent when tested with residual aqueous layer after solvent extraction of fresh whole garlic extract.