• 한국진공학회:학술대회논문집
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• 한국진공학회 2016년도 제50회 동계 정기학술대회 초록집
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• pp.375.1-375.1
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• 2016

Polymerase chain reaction (PCR) has revolutionized genetics and become one of the most popular techniques in modern biological and medical sciences. It can be used not only as an in vitro DNA amplification method but also used in many bioassay applications. The PCR can be used to exponentially produce a large number of DNA copies from a small quantity of DNA molecules in a few hours. However, as unwanted DNA fragments are also often manufactured, the amplification efficiency of PCR is decreased. To overcome this limitation, several nanomaterials have been employed to increase the specificity of the PCR reaction. Recently, graphene has attracted a great interest for its excellent electron transfer, thermal and biocompatibility. Especially, gold nanoparticle-coated graphene oxide (GO/AuNPs) led to enhance electron and thermal transfer rate and low-charge transfer resistance. Therefore, we report the development of a demonstration for the PCR efficiency using a large-scale production of the GO and combination of gold nanoparticles. Because a thermal conductivity is an important factor for improving the PCR efficiency in different DNA polymerases and different size samples. When PCR use GO/AuNPs, the result of transmission electron microscopy and real-time quantitative PCR (qPCR) showed an enhanced PCR efficiency. We have demonstrated that GO/AuNPs would be simply outperformed for enhancing the specificity and efficiency of DNA amplification procedure.

• 한국동물생명공학회지
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• 제34권4호
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• pp.259-266
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• 2019

Emulsion polymerase chain reaction (PCR) is performed on compartmentalized DNA, allowing a large number of PCR reactions to be carried out in parallel. Emulsion PCR has unique advantages in DNA amplification. It can be applied in many molecular biological assays, especially those requiring highly sensitive and specific DNA amplification. This review discusses the principle of emulsion PCR and its applications in biotechnology. Related technologies are also discussed.

• Fisheries and Aquatic Sciences
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• 제17권3호
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• pp.339-344
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• 2014

PCR amplification with universal primer is a useful tool for speciation of symbionts in marine eukaryote coupled with robust separation method such as denaturing high performance chromatography (DHPLC). To overcome the biased amplification, clamping PCR is recommended to suppress the amplification of host gene. In this study, we evaluated the efficiency of rare gene detection for two kinds of clamping probes which were successfully utilized for eukaryotic symbiont analysis: C3 linked nucleotide (C3) and peptide nucleic acid (PNA). PNA was 3-4 orders of magnitude higher than that of C3 tested in clamping efficiency and rare gene detection. This represented that PNA could be a more competent clamping probe for the enhancement of PCR amplification for rare symbiont genes.

• The Plant Pathology Journal
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• 제32권5호
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• pp.414-422
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• 2016

Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

• Bulletin of the Korean Chemical Society
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• 제29권2호
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• pp.497-498
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• 2008

• BMB Reports
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• 제28권4호
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• pp.359-362
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• 1995

The highly polymorphic variable number of tandem repeat (VNTR) loci in the human genome are informative markers for the genetic characterization of individuals in the paternity test and forensic science as well as for the study of human disease. In this study, VNTR loci D1S80 and D2S123 have been amplified by PCR and the amplified length polymorphic alleles were detected with a discontinuous vertical PAGE system and silver staining. For explicit DNA typing, PCR optimization, in which amplification efficiencies are similar over a wide range of allele sizes, non-specific amplifications are minimal, and new longer alleles have high amplification efficiency, has been performed by changing the PCR reaction buffer composition and thermal cycling conditions. It turned out that adding an appropriate amount of Tween 20 and NP40 to the PCR reaction buffer and raising the annealing temperature to $68^{\circ}C$ in thermal cycling made it possible for optimal VNTR loci amplification. A modified PAGE system for VNTR separation was established. Under these conditions, new longer alleles in the 01580 locus were discovered and 025123 pattern changes in colorectal tumors were observed. These technical tips are valuable for detecting various amplified fragment length polymorphisms.

• 생명과학회지
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• 제24권4호
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• pp.428-436
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• 2014

Loop-mediated isothermal amplification (LAMP)법은 등온에서 DNA 주형을 변성시키지 않고 실시하기 때문에, autocycling 가닥 변위 DNA 합성에 의존한다. 그래서 고가의 PCR 장비를 필요로 하지 않고 등온 유지가 가능한 저가의 장비인 항온 수조, 오븐, 온장고 등에서 증폭이 가능하다. 본 연구진은 Streptococcus parauberis의 random primer중에서 5개를 선정하여, 신장도가 높은 2개의 primer를 이용하여 최적 반응온도 및 최적 반응시간, 최적 반응 조건들을 확립하였다. 그리고 기존의 PCR과 LAMP의 민감도의 비교 분석을 측정한 결과, LAMP의 높은 검출 한계를 확인할 수 있었다. 본 논문에서는 non-target DNA의 영향을 받지 않고 등온 조건 하에서 DNA를 증폭시킬 수 있는 LAMP법과 SYBR-green I를 이용하여 시각화시켰으며, 기존의 PCR과 비교 분석함으로써, S. parauberis에 대한 신속하고 정확한 진단법을 확립하였다.

• 환경생물
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• 제22권2호
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• pp.329-335
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• 2004

To develop a biomarker for the monitoring of the contamination of estrogenic endocrine disrupters in the aquatic environment, reverse transcription -polymerase chain reaction (RT-PCR) analysis of vitellogenin (Vg) mRNA expression was optimized in Bombina orientalis, a Korean red bellied toad species. Based on partial cDNA sequences of both Vg and beta actin genes of B. orientalis, specific primers for RT-PCR of Vg and beta actin mRNAs were developed. Semiquantitative RT-PCR of the Vg mRNA in liver was optimized using a beta actin mRNA as an internal control in both sexes. In female RT-PCR using $1\;\mu{g}$ of the liver cDNA resulted in a linear increment in the PCR product of Vg from 18 to 34 cycles of amplification. In male, on the contrary, the RT- PCR product was first detected at 30 cycles of amplification and a linear increment was observed from 30 to 40 cycles of amplification, suggesting that male B. orientalis expresses minute amount of Vg mRNA which is a $2^{-12}$ equivalent of female. In conclusion, the optimized protocol for semiquantitative RT-PCR analysis of Vg mRNA level in B. orientalis male liver will be useful for the environmental monitoring the xenoestrogen contamination in the freshwater environment in Korea.

• 분석과학
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• 제29권1호
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• pp.43-48
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• 2016

법과학 분야에서 다양한 Y-STR 분석 키트가 개발되어 사용되고 있고, 새로운 키트의 법과학적 적용에 앞서 DNA 감정에 적절한 분석 키트들의 선정과 표준작업절차서의 작성을 위해 실험실 내의 내부적 유효성 검증 및 민감도 시험은 필수적인 과정이다. 본 논문에서는 새로운 상업용 키트인 Yfiler^® PLUS PCR Amplification Kit (Yfiler plus 키트, 2014년 출시)를 AmpF/STR^® Yfiler^TM PCR Amplification Kit (Yfiler 키트, 2004년 출시)와 비교함으로써 민감도에 대한 연구를 수행하였다. Yfiler plus 키트는 Yfiler 키트의 17 개 Y-STR 좌위를 포함하면서 새로운 10 개의 Y-STR 좌위가 추가되었다. 먼저, 표준 DNA 시료인 2800M, 007을 이용하여 두 키트 간의 민감도 차이를 분석하였고, 선별된 0.5 ng 미만의 법과학 현장시료 16 개로부터 검출률을 비교하였다. 그 결과, Yfiler 키트보다 Yfiler plus 키트가 높은 민감도와 검출률을 보였고, 더 많은 좌위에서 Y-STR 프로필을 얻을 수 있었다. 이러한 결과들로부터 낮은 농도의 법과학 현장시료에서 Yfiler plus 키트가 Y-STR 프로필을 검출하는데 더욱 효과적인 분석키트임이 확인되었다.

• 대한의생명과학회지
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• 제14권2호
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• pp.115-118
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• 2008

Salmonella typhi is frequent causes of foodborne illness and its detection is important for monitoring disease progression. In this study, by using general PCR and novel LAMP (Loop Mediated Isothermal Amplification) assay, we evaluated the usefulness of LAMP assay for detection of Salmonella typhi. In this LAMP assay, forward inner primer (FIP) and back inner primer (BIP) was specially designed for recognizing target invA gene. Target DNA was amplified and visualized as ladder-like pattern of bands on agarose gel within 60 min under isothermal conditions at $65^{\circ}C$. When the sensitivity and reproducibility of LAMP were compared to general PCR, there was no difference of reproducibility but sensitivity of LAMP assay was more efficient than PCR (the detection limit of LAMP assay was 30 fg, while the PCR assay was 3 pg). These results indicate that the LAMP assay is a potential and valuable means for detection of Salmonella typhi, especially for its rapidity, simplicity and low cost.