• 한국식품위생안전성학회지
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• 제23권2호
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• pp.121-128
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• 2008

본 연구는 Lightcycler (Roche)를 이용한 Real-Time PCR(LC-PCR)기법을 통하여 원유시료에서 신속, 정확하게 황색포도상구균을 검출하는 기법을 개발하고자 하였다. coagulase 전구체를 coding하는 113 bp의 coa 유전자의 증폭, melting curve 분석 및 DNA염기서열을 분석하여 황색포도상구균 특유의 유전자 검출하는 기법을 개발하였다. 또한 분리된 균주중 메치실린에 내성을 나타내는 균주를 검출하고자 penicillin-binding protein, PBP2a (mecA)를 coding 하는 209 bp의 mecA 유전자의 증폭, melting curve 분석 및 DNA염기서열을 분석하여 메치실린내성 황색 포도상구균을 real-time PCR 기법으로 검출하는 기술을 개발하였다. 본 실험에 따르면 647개의 원유시료중 6개의 시료에서 황색포도상구균이 검출되었으며 이중 2개의 시료에서 분리된 황생포도상구균이 메치실린내성 황색포도상구균임을 확인하였다. 또한 DNA 검출한계는 10 fg으로 기존 PCR에 비해 매우 감도가 우수한 것을 확인하였다. 또한 3개의 원유시료에서 돼지나 소의 삼출성 피부염의 원인균인 Staphylococcus chromogenes가 분리되었다.

• 통합자연과학논문집
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• 제5권3호
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• pp.163-167
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• 2012

UV spectrophotometer was used to detect protein-DNA complex from DNA melting profile under constant temperature increase. Melting temperature (Tm) was $43^{\circ}C$ in copA duplex DNA alone. In the presence of Proteus mirabilis transcription regulator protein (PMTR) protein at 0.2 and 0.4 ${\mu}M$, Tm's were $45{\pm}0.5$ and $47.6{\pm}0.6^{\circ}C$, respectively. According to fluorescence polarization and gel shift assay. PMTR:copA complex was detected by the retarded migration on gel and the dissociation constant ($K_d$) was $(9.2{\pm}2.8){\times}10^{-9}M$.

• Asian Pacific Journal of Cancer Prevention
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• 제14권11호
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• pp.6619-6623
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• 2013

Background:The aim of the research was to explore a cost effective, fast, easy to perform, and sensitive method for epidermal growth factor receptor (EGFR) mutation testing. Methods: High resolution melting analysis (HRM) was introduced to evaluate the efficacy of the analysis for dectecting EGFR mutations in exons 18 to 21 using formalin-fixed paraffin-embedded (FFPE) tissues and plasma free DNA from 120 patients. Results: The total EGFR mutation rate was 37.5% (45/120) detected by direct sequencing. There were 48 mutations in 120 FFPE tissues assessed by HRM. For plasma free DNA, the EGFR mutation rate was 25.8% (31/120). The sensitivity of HRM assays in FFPE samples was 100% by HRM. There was a low false-positive mutation rate but a high false-negative rate in plasma free DNA detected by HRM. Conclusions: Our results show that HRM analysis has the advantage of small tumor sample need. HRM applied with plasma free DNA showed a high false-negative rate but a low false-positive rate. Further research into appropriate methods and analysis needs to be performed before HRM for plasma free DNA could be accepted as an option in diagnostic or screening settings.

• 미생물학회지
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• 제28권1호
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• pp.19-26
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• 1990

To examine the effects of sequence variations near the transcriptional start site on the rate of formation of the open complexes at bacteriophage $\lambda P_{R}$ promoter, two mutant promoters were created by site-specific mutagenesis using synthetic oligonucleotides. Mutant I coatains changes at positions -3 and -4 from TT to CC, thus having a 6-bp long G/C stretch between -10 region and transciptional start site (+1). Mutant II has changes at positions -5 and -6 from GG to AA, thereby having a 9-bp long A/T stretch between positions -11 and -3. Selective filter binding assays were performed to measure the rate of formation of the open complexes between the wild-type or two mutant $P_{R}$ promoters on 664 bp fragments and E. coli RNA polymerase at two temperatures. At 37.deg.C, the wild-type and two mutants showed similar rates for the formation of open complex. The second order rate constant $k_{a}$ and $\tau _{int}$, as determined from the .tau.-plot analysis, were $(6.0\pm0.4)\times10^{6}M^{-1}sec^{-1}$ and $11\pm5$sec, respectively. At 18.deg.C, however, the wild-type and two mutant promoters showed differences in the kinetic parameters. k for the wild-type promoter was (2.2$\pm$0.1)\times 10^{6}M^{-1}sec^{-1}$ and $\tau _{int}$ was 76$\pm$sec. Mutant I and II exhibited differences mainly in the rate of isomerization ($\tau_{int,I}=91\pm$10 sec, int,II=34$\pm$ sec), whereas the second order rate constant $k_{a}$ was similar to the wild type value. This result implies that at $18^{\circ}C$, the isomerization rate is determined by both protein conformational change and DNA melting, which are separable kinetically according to the 3-step mechanism of Roe et al.(1984,1985), and that the base changes affected mainly the rate of DNA melting as predicted.lting as predicted.

• 환경생물
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• 제40권2호
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• pp.199-205
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• 2022

Two tree frogs, Dryophytes suweonensis and Dryophytes japonicus, inhabiting Korea, are morphologically similar and share the same habitats. Therefore, they are identified mainly through their calls, especially for males. Dryophytes suweonensis is registered as an endangered (IUCN: EN grade) and protected species in South Korea. Thus, it is necessary to develop a method to rapidly identify and discriminate the two species and establish efficient protection and restoration plans. We identified significant genetic variation between them by sequencing a maternally-inherited mitochondrial 12S ribosomal DNA region. Based on the sequence data, we designed a pair of primers containing 7bp differences for high resolution melting(HRM) analysis to rapidly and accurately characterize their genotypes. The HRM analysis using genomic DNA showed that the melting peak for D. suweonensis was 76.4±0.06℃, whereas that of D. japonicus was 75.0±0.05℃. The differential melt curve plot further showed a distinct difference between them. We also carried out a pilot test for the application of HRM analysis based on immersing D. suweonensis in distilled water for 30 min to generate artificial environmental DNA(eDNA). The results showed 1.10-1.31℃ differences in the melting peaks between the two tree frog samples. Therefore, this HRM analysis is rapid and accurate in identifying two tree frogs not only using their genomic DNA but also using highly non-invasive eDNA.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.539-542
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• 2002

We propose new encoding method for numerical data in DNA using temperature gradient. To represent numerical values in DNA sequences, we introduce melting temperature. Since DNA strands representing smaller values have a lower Tm, they tend to denature with ease and also easily amplified by denaturation temperature gradient PCR. We also implement a local search molecular algorithm using temperature gradient, which is contrasted to conventional exhaustive search molecular algorithms. The proposed methods are verified by solving an instance of the travelling salesman problem. We could effectively amplify the correct solutions and the use of temperature gradient made the detection of solutions easier.

• Journal of Applied Biological Chemistry
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• 제49권4호
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• pp.143-147
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• 2006

A rapid PCR-based assay for detecting hepatitis B viral DNA(HBV DNA) in serum and plasma was developed using a new PCR instrument named GenSpector(TMC-1000, Samsung electronics). PCR was carried out using a chip-based platform, which enabled 50 PCR cycles with internal controls, and melting-curve analysis in 30 minutes. Verification of the amplified HBV DNA product and the internal control was based on specific melting temperatures(Tm) analysis, executed by the GenSpector software. Primers were designed within the region conserved through HBV genotypes A to F. The lower limit of detection was 840 copies/ml serum, conducted with serial dilutions of a HBV DNA positive control(ACCURUN 325 series 700, Boston Biomedica Inc.). The assay was also compared to another assay for HBV DNA(Versant HBV DNA 3.0 assay, Bayer HealthCare) for 200 samples(each 100 clinical negative and positive samples). The sensitivity and specificity were 100% matched. This rapid PCR-based assay is specific, reproducible, and enables qualitative detection of HBV 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.

• Bulletin of the Korean Chemical Society
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• 제31권12호
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• pp.3794-3796
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• 2010

• 한국식품위생안전성학회지
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• 제26권2호
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• pp.114-119
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• 2011

본 연구에서는 신선편의 채소와 과일에서 주로 검출되는 대장균(E. coli Ol577:H7), 리스테리아(L. monocytogenes), 살모넬라(Salmonella, spp.), 형색포도상구균(S. aureus)을 검출하고 동정할 수 있는 real time PCR 방법을 melting curve 분석을 활용하여 single tube 반응으로 두 종의 식중독균을 동시 검출하는 방법을 개발하고자 하였다. 대장균의 ${\beta}$-glucuronidase (uidA), 황색포도상구균의 thermonuclease (nuc), 리스테리아의 hemolycin (hly), 살모넬라의 tetrahionate reductase (ttr) 를 특이적으로 검출할 수 있는 4종의 프라이머 세트에 대한 real-time PCR의 melting curve 분석을 통하여 황색포도상구균과 대장균 동시분석 시 $T_m$ 값이 $80.6{\pm}0.9^{\circ}C$와 $86.9{\pm}0.5^{\circ}C$, 리스테리아와 살모넬라 동시분석 시 Tm 값이 $80.4{\pm}0.6^{\circ}C$와 $88.1{\pm}0.11^{\circ}C$로 확인하였고, 그 결과 정제되어진 각 식중독균의 genomic DNA를 주형으로 한 duplex real-time PCR 방법이 uniplex real-time PCR 방법과 마찬가지로 10 genome equivalents 까지 검출할 수 있는 민감도를 나타내었다. 또한, 양배추에 네 종의 식중독균을 접종하고, 증균배양 없이 DNA를 추출하여 duplex real-time PCR 을 수행한 결과 모든 식종목균에서 $10^3$ CFU/g의 검출 한계를 나타내었다. 결과적으로 개발된 melting curve 분석을 이용한 duplex real-time PCR 방법은 식품안전 증진을 위한 시간, 노동력, 비용 절감에 있어서 유효한 방법이 될 것으로 판단된다.