• Journal of Microbiology and Biotechnology
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• v.20 no.10
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• pp.1463-1470
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• 2010

E. coli has long been widely used as a host system for the manufacture of recombinant proteins intended for human therapeutic use. When considering the impurities to be eliminated during the downstream process, residual host cell DNA is a major safety concern. The presence of residual E. coli host cell DNA in the final products is typically determined using a conventional slot blot hybridization assay or total DNA Threshold assay. However, both the former and latter methods are time consuming, expensive, and relatively insensitive. This study thus attempted to develop a more sensitive real-time PCR assay for the specific detection of residual E. coli DNA. This novel method was then compared with the slot blot hybridization assay and total DNA Threshold assay in order to determine its effectiveness and overall capabilities. The novel approach involved the selection of a specific primer pair for amplification of the E. coli 16S rRNA gene in an effort to improve sensitivity, whereas the E. coli host cell DNA quantification took place through the use of SYBR Green I. The detection limit of the real-time PCR assay, under these optimized conditions, was calculated to be 0.042 pg genomic DNA, which was much higher than those of both the slot blot hybridization assay and total DNA Threshold assay, where the detection limits were 2.42 and 3.73 pg genomic DNA, respectively. Hence, the real-time PCR assay can be said to be more reproducible, more accurate, and more precise than either the slot blot hybridization assay or total DNA Threshold assay. The real-time PCR assay may thus be a promising new tool for the quantitative detection and clearance validation of residual E. coli host cell DNA during the manufacturingprocess for recombinant therapeutics.

• Environmental Mutagens and Carcinogens
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• v.17 no.2
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• pp.71-77
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• 1997

The single cell gel electrophoressis(SCGE) assay, also known as the comet assay, is a rapid, simple, visual and sensitive technique for measuring and analysing DNA breakage in mammalian cells. The SCGE or comet assay is a promising test for the detection of DNA damage and repair in individnal cells. It has widespread potential applications in DNA damage and repair studies, genotoxicity testing and biomonitoring. In this microgel electrophoresis technique, cells are embedded in agarose gel on microscope slides, iysed and electrophoresed under alkaline conditions. Cells with increased DNA damage display increased migration of DNA from the nucleus towards the anode. The length of DNA migration indicates the amount of DNA breakage in the cell. The comet assay is also capable of identifying apoptotic cells which contain highly fragmented DNA. Here we review the development of the SCGE assay, existing protocols for the detection and analysis of comets, the relevant underlying principles determining the behaviour of DNA and the potential applications of the technique.

• Journal of Life Science
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• v.8 no.1
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• pp.67-71
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• 1998

In order to develop new antitumor agents from fermentation broths, we used cccDNA breakage assay abd sulforhodamine B assay for prescreening. As a result, it was shown that sample reach 3.3% when using cccDNA breakage assay. In sulforhodamine B assay, we obtained 4 acive fraction against A549 (a cell line of human lung carcinoma) and SK-OV-3 (a cell line of human adenocarcinoma). These results suggest that these assay would be a promising method for antitumor prescreening from microbial sources.

• Journal of Nutrition and Health
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• v.35 no.2
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• pp.213-222
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• 2002

The alkaline comet assay has been used with increasing popularity to investigate the level of DNA damage in biomonitoring studies within the last decade in Western countries. The purpose of this study was to evaluate the usefulness of the alkaline comet assay as a biomarker of oxidative DNA damage for monitoring in the Korean population, and also to evaluate the effect of nutritional status and lifestyle factors on H2O2 induced oxidative DNA damage measured by the alkaline comet assay in human lymphocytes. The study population consisted of 61 healthy Korean male volunteers, aged 20-28. Epidemiological background data including dietary habits, smoking habits and anthropometrical measurements were collected through personal interviews. After blood collection, the comet assay in peripheral lymphocytes and plasma lipids analysis was carried out and the results analyzed. Tail moment (TM) and tail length (TL) of the comet assay were use\ulcorner to measure DNA damage in the lymphocytes of the subjects. Statistically significant (p < 0.05) positive correlations were observed between DNA damage (TM or TL) and smoking habits expressed as cigarettes smoked per day and pack years (r = 0.311 and 0.382 for TM, r = 0.294 and 0.350 for TL, respectively). There were also significant positive correlations between DNA damage parameter and waist-hip ratio. Higher plasma triglyceride levels were associated with increased damage to DNA. There were no correlations between the consumption frequencies of vegetables and DNA damage to the subjects. However, consumption frequencies of fruit and fruit juice intake were inversely associated with the TM and TL. The results indicate that die comet assay is a simple, rapid and sensitive method for detecting lymphocyte DNA damage induced by cigarette smoking. Consumption of fruit or fruit juices could potentiall modify the damaged DNA in the human peripheral lymphocytes of young Korean men.

• Journal of the Korean Electrochemical Society
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• v.7 no.1
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• pp.44-48
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• 2004

The two-component type enzyme amplified amperometric DNA assay is described to use an ambient $O_2$ of the substrate of the DNA labeling enzyme. Although the assay detects DNA only at > 0.5M concentration, a concentration $\~10^6$ fold higher than the sandwich-type enzyme amplified amperometric DNA assay, it can be run with an always available substrate. The assay utilizes screen-printed carbon electrodes (SPEs) which were pre-coated by a co-electrodeposited film of an electron conducting redox hydrogel and a 37-base long single-stranded DNA sequence. The DNA in the electron conducting film hybridizes and captures, when present, the 37-base long detection-DNA, which is labeled with bilirubin oxidase (BOD), an enzyme catalyzing the four-electron reduction of $O_2$ to water. Because the redox hydrogel electrically connects the BOD reaction centers to the electrode, completion of the sandwich converts the film from non-electrocatalytic to electrocatalytic for the reduction of $O_2$ to water when the electrode is poised at 200 mV vs. Ag/hgCl. The advantage or the assay over the earlier reported sandwich type enzyme amplified amperometric DNA assay, in which the amplifying enzyme was horseradish peroxidase, is that it utilizes ambient $O_2$ instead of the less stable and naturally unavailable $H_2O_2$.

• Korean Journal of Plant Resources
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• v.22 no.3
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• pp.195-202
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• 2009

Schisandra chinensis have been traditionally used in Asia for the treatment of dyspnea, cough, mouth dryness, spontaneous diaphoresis, nocturnal diaphoresis, nocturnal emission, dysentery, insomnia and amnesia. The purpose of this study is to evaluate the protective effects of Schisandra chinensis on oxidative DNA damage and lipid peroxidation induced by ROS in non cellular and cellular system. DPPH radical, hydroxyl radical and hydrogen peroxide scavenging assay were used to measure the antioxidant activities. Phi X-174RF I plasmid DNA cleavage assay and intracellular DNA migration assay were used to evaluate the protective effect on oxidative DNA damage. MTT assay and lipid peroxidation assay were used for evaluating the protective effect on oxidative cell damage. It was found to scavenge DPPH radical, hydrogen peroxide and hydroxyl radical and it inhibited oxidative DNA damage, lipid peroxidation and cell death induced by hydroxyl radical. These data indicate that Schisandra chinensis possesses a spectrum of antioxidant and DNA-protective properties

• Environmental Mutagens and Carcinogens
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• v.21 no.1
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• pp.34-43
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• 2001

Di-2-ethylhexyl phthalate (DEHP) is the most commonly used phthalate ester in polyvinyl chloride formulations including food packing and storage of human blood. DEHP is a well known as non-genotoxic carcinogen and endocrine disrupting chemical (EDC). DEHP have shown all negative results in ICH-guildeline recommended standard genotoxicity test battery. In this study, to assess the clastogenic and DNA damaging effect in human-derived tissue specific cells, DEHP was treated in human derived MCE-7 cells, HepG2 cells, LNCap cells, BeWo cells, MCE-10A cells, and female peripheral blood cells using micronucleus assay and in human breast carcinoma MCF-7 cells up to $1.28$\times$10^{-2}$ M using Comet assay. The in vitro micronucleus assay is a mutagenicity test system for the detection of chemicals which induce the formation of small membrane bound DNA fragment i.e. micronuclei in the cytoplasm of interphase cells, originated from clastogenic and/or aneugenic mechanism. The single cell gel electrophoresis assay (Comet assay) is used to detect DNA strand-breaks and alkaline labile site. In our results, DEHP increased significantly and/or dose-depentently and time-dependently micronucleus frequency at the 6 and 24 hr without metabolic activation system only in MCE-7 cells. DEHP treated with 2 hrs in MCF-7 cells using Comet assay induced DNA damage dose-depentantly.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.36 no.4
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• pp.346-351
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• 2003

Single-cell gel electrophoresis (comet assay) was used to detect DNA single strand break in blood cells from several marine fish species. Three fish species were collected from Georgia coastal area. Mummichog, Fundulus heteroclitus showed higher DNA damage than sea bass, Lateolabrax japonicus and trout, Oncorhynchus masou masou under the same experimental conditions. Mummichogs had more alkaline-labile sites on their DNA than other fish species. The comet assay with mummichog blood cells at pH 12.5 showed a dose-response curve with the increasing concentrations of hydrogen peroxide. While the isolated leucocytes showed no increase of DNA damage after in vitro exposure to 2-methyl-1,4-naphthoquinone (MNQ), erythrocytes showed dose-dependent DNA damage. These results indicate that the comet assay can be applied successfully as a bioassay using erythrocyte for environmental monitoring.

• Journal of Applied Biological Chemistry
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• v.49 no.4
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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.

• Journal of the Korean Society of Food Science and Nutrition
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• v.37 no.4
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• pp.472-476
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• 2008

The possibility of DNA comet assay as a rapid method for screening the irradiated vegetables and grains was evaluated. Vegetables (spring onion, garlic, and tomato) irradiated at $0{\sim}3$ kGy and grains (rice flour and black soybean) irradiated at $0{\sim}9$ kGy were used as samples. Optimum DNA comet assay conditions, such as elution, sedimentation of suspension, and lysis time of cell, were established. The optimum conditions for vegetables were 10 min for the elution time, 0 min for the sedimentation time, and 5 min for the lysis time. The optimum conditions for grains were 15 min for the elution time, 60 min for the sedimentation time, and 30 min for the lysis time. For the food application of DNA comet assay, it was possible to screen various food samples irradiated at the following doses: spring onion at 2 kGy, garlic at 3 kGy, tomato at 1 kGy, rice flour at 9 kGy, and black soybean at 3 kGy. Each sample showed different forms and sizes in DNA comet. Therefore, further studies on various methods using comet shape, concentration, or area in DNA comet assay are necessary.