• Title/Summary/Keyword: Emulsion PCR

Search Result 13, Processing Time 0.026 seconds

Principle of Emulsion PCR and Its Applications in Biotechnology

  • Chai, Changhoon
    • Journal of Animal Reproduction and Biotechnology
    • /
    • v.34 no.4
    • /
    • pp.259-266
    • /
    • 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.

Emulsion PCR Improves the Specificity and Sensitivity of PCR-based Pathogen Detection (식중독균 검출의 민감도 향상을 위한 Emulsion PCR 적용)

  • Chai, Changhoon
    • Journal of Dairy Science and Biotechnology
    • /
    • v.34 no.1
    • /
    • pp.43-49
    • /
    • 2016
  • Emulsion PCR (ePCR) has recently gained interest in the areas of food safety and biotechnology owing to its highly specific and sensitive performance in the amplification of target DNA. To facilitate the applications of ePCR to food safety and biotechnology, this paper describes the principles of ePCR and the factors that should be considered in designing ePCR. In addition, current research and applications related to ePCR are discussed.

Detection of rare point mutation via allele-specific amplification in emulsion PCR

  • Cheng, Changming;Zhou, Yin;Yang, Chao;Chen, Juan;Wang, Jie;Zhang, Jie;Zhao, Guoping
    • BMB Reports
    • /
    • v.46 no.5
    • /
    • pp.270-275
    • /
    • 2013
  • It is essential to analyze rare mutations in many fields of biomedical research. However, the detection of rare mutations is usually failed due to the interference of predominant wild-type DNA surrounded. Herein we describe a sensitive and facile method of detecting rare point mutation on the basis of allele-specific amplification in emulsion PCR. The identification and selective amplification of rare mutation are accomplished in one-pot reaction. The allele-specific primers coupled on magnetic beads allow the exclusive amplification and enrichment of the mutant amplicons. The productive beads bearing mutant amplicons are subsequently stained with the fluorescent dyes. Thus, the rare point mutations with a percentage as low as 0.1%, can be detected by fluorescent analysis. The relative percentages of mutation among different samples can be roughly accessed by counting the fraction of fluorescent positive beads through flow cytometry.

Enhancement of Emulsion-mediated Gene Expression by Using Chitosan as a Pre-Condensing Agent

  • Chun, Soo-Kyung;Choi, Sung-Hee;Kim, Adele;Ahn, Woong-Shick;Kim, Chong-Kook
    • Proceedings of the PSK Conference
    • /
    • 2002.10a
    • /
    • pp.426.3-427
    • /
    • 2002
  • The aim of this study was to enhance the transfection efficiency of emulsion-mediated gene expression by using chitosan, Conventional DNA/emulsion complexes and precondensed DNA/emulsion complexes were prepared by adding either naked or precondensed plasmids to cationic emulsion. The zeta potential. TEM, and size of transfection complexes were measured. In vitro transfection efficiency for boty complexes was also studied by several methods: flow cytometer, expression analysis by confocal microscope, RT-PCR, and in addition. (omitted)

  • PDF

Recent advances in microfluidic technologies for biochemistry and molecular biology

  • Cho, Soong-Won;Kang, Dong-Ku;Choo, Jae-Bum;Demllo, Andrew J.;Chang, Soo-Ik
    • BMB Reports
    • /
    • v.44 no.11
    • /
    • pp.705-712
    • /
    • 2011
  • Advances in the fields of proteomics and genomics have necessitated the development of high-throughput screening methods (HTS) for the systematic transformation of large amounts of biological/chemical data into an organized database of knowledge. Microfluidic systems are ideally suited for high-throughput biochemical experimentation since they offer high analytical throughput, consume minute quantities of expensive biological reagents, exhibit superior sensitivity and functionality compared to traditional micro-array techniques and can be integrated within complex experimental work flows. A range of basic biochemical and molecular biological operations have been transferred to chip-based microfluidic formats over the last decade, including gene sequencing, emulsion PCR, immunoassays, electrophoresis, cell-based assays, expression cloning and macromolecule blotting. In this review, we highlight some of the recent advances in the application of microfluidics to biochemistry and molecular biology.

Detection and quantitation of Bacillus cereus, Staphylococcus aureus, Salmonella Typhimurium and Escherichia coli O157:H7 by droplet digital PCR (Droplet Digital PCR을 이용한 Bacillus cereus, Staphylococcus aureus, Salmonella Typhimurium과 Escherichia coli O157:H7의 검출 및 정량)

  • Kim, Jin-Hee;Yoon, JinSun;Lee, Da-Young;Kim, Dongho;Oh, Se-Wook
    • Korean Journal of Food Science and Technology
    • /
    • v.48 no.5
    • /
    • pp.454-460
    • /
    • 2016
  • In this study, we investigated the possibility of Droplet digital PCR (ddPCR) for detection of foodborne pathogens. ddPCR combines partitioning of PCR reactions into several thousands or millions of individual droplets in a water-oil emulsion, and counting of positive PCR reaction using flow cytometry. Four species of foodborne pathogens, Bacillus cereus, Staphylococcus aureus, Salmonella Typhimurium and Escherichia coli O157:H7, were used to quantify the target sequence with each of the designed primers and double stranded DNA-binding Evagreen dye. All tested foodborne pathogens showed a detection limit ranging from $100fg/{\mu}L$ to $10ng/{\mu}L$. It was concluded that ddPCR could be used to detect very low concentrations of foodborne pathogens from complex food matrices. For multi-detection of target pathogens, we also tested the samples using multiplex ddPCR and obtained successful results.

Antioxidant and anti-aging effects of Alpinia galanga L. rhizome extracts and preservation of antioxidant effects in W/O type emulsion (갈랑가 뿌리 추출물의 항산화, 항노화 효과 및 W/O형 에멀젼에서 항산화 효과의 보존성)

  • Sun Young Yoon;Bong Hwan Kim;Young Ah Jang;Se Gie Kim
    • Journal of Applied Biological Chemistry
    • /
    • v.66
    • /
    • pp.424-435
    • /
    • 2023
  • As a result of this study, DPPH radical scavenging activity was 81.8% at a concentration of 100 ㎍/mL of Alpinia galanga L. rhizome 70% ethanol extract (AG.E), and ABTS+ radical scavenging activity was confirmed to be 99.8%, similar to L-Ascorbic acid (AA), at a low concentration of 50 ㎍/mL AG.E. To measure anti-aging activity, collagenase and elastase inhibitory activities were measured and AG.E showed higher inhibitory effects than epigallocatechin gallate (EGCG) starting at a low concentration of 50 ㎍/mL. In particular, AG.E showed inhibitory effects more than three times that of EGCG at a concentration of 500 ㎍/mL. In order to verify anti-aging effect of AG.E in CCD-986sk cell, good anti-aging effect was obtained in various experiments stimulated with UVB. In a gene expression analysis experiment using RT-PCR, the COL1A mRNA expression level was found to increase 2.90 times compared to no addition at a low concentration of 20 ㎍/mL AG.E, confirming the possibility of developing it as a good functional material related to anti-aging. As a basic study on temporal biological activity preservation ratio of material when applied to formulations, AG.E, and AA were added to a stable W/O type emulsion and stored in a thermostat at 25 ℃ for 60 days. As a result of measuring DPPH and ABTS+ radical scavenging activities on the 1st, 30th, and 60th days, it was confirmed that antioxidant effects are maintained at a high level over time in formulations.

Development of Rapid Diagnostic Technology for Pig Disease (2) - Rapid detection of PPE in the pig feces -

  • Kim, Hyuck-Joo;Hong, Jong-Tae;Yu, Byeong-Kee;Kim, Giyoung;Kim, Suk
    • Journal of Biosystems Engineering
    • /
    • v.38 no.2
    • /
    • pp.121-128
    • /
    • 2013
  • Purpose: Porcine proliferative enteropathy (PPE), caused by the obligate intracellular bacterium Lawsonia intracellularis, is a widely distributed disease throughout the world causing substantial economic loss. In order to diagnose PPE rapidly, the rapid kit was developed and tested. Methods: In this study, a rapid kit was developed to screen the PPE rapidly at the pig farm. Also, occult blood test with fecal occult blood (FOB) kit was done for detecting the blood in pig feces which might be the evident of hemorrhagic PPE. For developing the kit, we tested fecal samples of PPE infected pigs diagnosed by polymerase chain reaction (PCR) method. Results: With the developed rapid kit, Lawsonia intracellularis was detected in high density emulsion of ileum. On the other hand, the test result of detecting Lawsonia in feces showed too high non-specific response. In addition, nevertheless the FOB test result showed that blood evident could be founded in pig feces, the diagnosing result was not fit to PCR test result, which shows blood in pig feces could be from not only hemorrhagic PPE but also many reasons. Conclusions: To deal with the PPE effectively, it will be better for farmers to screen the PPE in earlier stage with easy and rapid diagnosing tool on farm. This study found out that the rapid kit could detect the Lawsonia intracellularis and hemoglobin in pig feces. However, the non-specific response to negative samples of PPE was too high to use at a pig farm. Further research is needed for lowering the non-specific response with the rapid kit.

Cold Shock Response of an Antarctic Streptomyces Strain Showing Demulsifying Ability

  • Lee Yoo Kyung;Kim Hyo Won;Hyun Kwang Soon;Lee Hong Kum
    • Proceedings of the Microbiological Society of Korea Conference
    • /
    • 2001.11a
    • /
    • pp.138-145
    • /
    • 2001
  • The hydrophobic spores of Streptomyces sp. AA8321 isolated from the Antarctic coast displayed demulsification ability. The aerial spores demulsified an emulsion of kerosene/$0.2\%$ Triton X-100 (2:1, v/v) to $50\%$ and $95\%$ within 1 min contact at the concentrations of $5.0{\times}10^7$ and $1.0{\times}10^8$ spores/ml, respectively. A cold shock protein (csp) gene was cloned from the hydrophobic spore- producing Streptomyces sp. AA8321 using PCR. It encoded a low molecular protein with 68 amino acids showing very low homology with previously reported csp genes. Only the sequence of the first six amino acids was just the same and yet others were different. RNA blot analysis indicated that the csp gene was induced by cold shock, i.e., transferring from $30^{\circ}C$ to $10^{\circ}C$, and this cold shock response proposed that the isolated gene be a new type of csp gene.

  • PDF

Comparison of Inhibitory Effect of 17-DMAG Nanoparticles and Free 17-DMAG in HSP90 Gene Expression in Lung Cancer

  • Mellatyar, Hassan;Akbarzadeh, Abolfazl;Rahmati, Mohammad;Ghalhar, Masoud Gandomkar;Etemadi, Ali;Nejati-Koshki, Kazem;Zarghami, Nosratallah;Barkhordari, Amin
    • Asian Pacific Journal of Cancer Prevention
    • /
    • v.15 no.20
    • /
    • pp.8693-8698
    • /
    • 2014
  • Background: Up-regulation of hsp90 gene expression occurs in numerous cancers such as lung cancer. D,L-lactic-co-glycolic acid-poly ethylene glycol-17-dimethylaminoethylamino-17-demethoxy geldanamycin (PLGA-PEG-17DMAG) complexes and free 17-DMAG may inhibit the expression. The purpose of this study was to examine whether nanocapsulating 17DMAG improves the anti cancer effect over free 17DMAG in the A549 lung cancer cell line. Materials and Methods: Cells were grown in RPMI 1640 supplemented with 10% FBS. Capsulation of 17DMAG is conducted through double emulsion, then the amount of loaded drug was calculated. Other properties of this copolymer were characterized by Fourier transform infrared spectroscopy and H nuclear magnetic resonance spectroscopy. Assessment of drug cytotoxicity on the grown of lung cancer cell line was carried out through MTT assay. After treatment, RNA was extracted and cDNA was synthesized. In order to assess the amount of hsp90 gene expression, real-time PCR was performed. Results: In regard to the amount of the drug load, IC50 was significant decreased in nanocapsulated(NC) 17DMAG in comparison with free 17DMAG. This was confirmed through decrease of HSP90 gene expression by real-time PCR. Conclusions: The results demonstrated that PLGA-PEG-17DMAG complexes can be more effective than free 17DMAG in down-regulating of hsp90 expression by enhancing uptake by cells. Therefore, PLGA-PEG could be a superior carrier for this kind of hydrophobic agent.