• Title/Summary/Keyword: Slot Hybridization

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Quantitative Detection of Residual E. coli Host Cell DNA by Real-Time PCR

  • Lee, Dong-Hyuck;Bae, Jung-Eun;Lee, Jung-Hee;Shin, Jeong-Sup;Kim, In-Seop
    • 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.

Microbial Communities of Activated Sludge in an Anaerobic/Aerobic Sequencing Batch Reactor using Slot Hybridization (Slot Hybridization을 이용한 연속 회분식 반응기내 미생물 분포 조사)

  • Jeon, Che Ok;Shin, Kum-Joo;Lee, Dae Sung;Suh, Pann-Ghill;Park, Jong Moon
    • Journal of Korean Society of Environmental Engineers
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    • v.22 no.5
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    • pp.939-947
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    • 2000
  • Enhanced biological phosphorus removal (EBPR) was performed in an anaerobic/aerobic sequencing batch reactor (SBR). Influent was a synthetic wastewater based on acetate as a carbon source. The sludge age and hydraulic retention time were kept at 10 days and 16 hrs, respectively, Phosphate release during the anaerobic period and phosphate uptake in aerobic period were increased gradually with time. and after about 200 days, steady-state operation could be achieved with complete removal of influent phosphate. Number distribution of microbial community in the sludge performing EBPR was investigated during the steady state operation. 17 rRNA targeted oligonucleotide probes were designed and slot hybridization technique was used to determine the number distribution of each microorganism. In the acetate fed SBR, rRNA belonging to the beta subclass of proteobacteria was the most dominant in total rRNA and rRNA matching to CTE probe was the second, rRNAs of Acinetobacter, Aeromonas and Pseudomonas, which are usually thought as phosphorus accumulating organisms in EBPR processes, constituted less than 10% of total rRNA. From this community analysis, it was inferred that microorganisms belong to the beta subclass of proteobacteia (BET) and CTE such as Rhodocyclus group were important in biological phosphorus removal. Therefore, the role of Acinetobacter, Aeromonas and Pseudomonas in the EBPR might have been overestimated.

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Chemoprotective Effect of Methanol Extract of Hedera rhombea Loaves on the Reversal of Cytochrome P-450 Activities Induced by Carbon Tetrachloride (사염화탄소로 유도된 Cytochrome P-450 활성도의 전환으로 본 Hedera rhombea 잎의 메탄올 추출물의 간독성 감소작용)

  • 홍영숙;김형래;배영숙;박상신
    • Biomolecules & Therapeutics
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    • v.3 no.4
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    • pp.245-250
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    • 1995
  • The carbon tetrachloride($CCl_4$) has been demonstrated to have a hepatotoxic effect in human or many other species. To investigate the enzyme induction of mixed function oxygenases in liver of male Sprague-Dawley rats a single 0.1, 0.5 mι/kg dose of carbon tetrachloride were given. At 24 hr after a single dose of 0.1 mι CC1$_4$/kg weight, methanol extract of Hedera rhombea leaves was administered with 100, 500 mg/kg weight. Assays of 7-ethoxyresorufin-Ο-deethylation(EROD),7-benzyloxyresorufin-Ο-deathylation(BROD),4-nitro-phenol-UDP-glucuronosyltransferase(UDPGT), Western blot and RNA slot blot were used as representatives of the activities of cytochrome P-450 enzymes. The change of the activity of CYP1A1 form measured by EROD assay and Western analysis using 1-7-1 monoclonal antibody was not observed. The activity CYP2B1 form by BROD assay and using 2-66-3 monoclonal antibody was remarkably increased. Elevated level of CYP2B1 mRNA was shown by slot hybridization with 2B1-specific probe. Administration of methanol extract of Hedera rhombea leaves reversed the enzyme activity and the level of mRNA, which suggest the chemoprotective effect of methanol extracts of Hedera rhombea leaves to carbon tetrachloride hepatotoxlcity.

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Upregulated expression of the cDNA fragment possibly related to the virulence of Acanthamoeba culbertsoni

  • Im, Kyung-Il;Park, Kwang-Min;Yong, Tai-Soon;Hong, Yong-Pyo;Kim, Tae-Eun
    • Parasites, Hosts and Diseases
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    • v.37 no.4
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    • pp.257-263
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    • 1999
  • Identification of the genes responsible for the recovery of virulence in brain-passaged Acanthamoeba culbertsoni was attempted via mRNA differential display polymerase chain reaction (mRNA DD-PCR) analysis. In order to identify the regulatory changes in transcription of the virulence related genes by the brain passages, mRNA DD-PCR was performed which enabled the display of differentially transcribed mRNAs after the brain passages. Through mRNA DD-PCR analysis. 96 brain-passaged amoeba specific amplicons were observed and were screened to identify the amplicons that failed to amplify in the non-brain-passaged amoeba mRNAs. Out of the 96 brain-passaged amoeba specific amplicons, 12 turned out to be amplified only from the brain-passaged amoeba mRNAs by DNA slot blot hybridization. The clone, A289C, amplified with an arbitrary primer of UBC #289 and the oligo dT$_{11}$-C primer, revealed the highest homology (49.8%) to the amino acid sequences of UPD-galactose lipid transferase of Erwinia amylovora, which is known to act as an important virulence factor. The deduced amino acid sequences of an insert DNA in clone A289C were also revealed to be similar to cpsD, which is the essential gene for the expression of type III capsule in group B streptococcus. Upregulated expression of clone A289C was verified by RNA slot blot hybridization. Similar hydrophobicity values were also observed between A289C (at residues 47-66) and the AmsG gene of E. amylovora (at residues 286-305: transmembrane domains). This result suggested that the insert of clone A289C might play the same function as galactosyl transferase controlled by the AmsG gene in E. amylovora.a.

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Antiviral Effects of Natural Products on the Inhibition of Hepatitis B Virus DNA Replication in 2.2.15 Cell Culture System

  • Nam, Kung-Woo;Chang, Il-Moo;Choi, Jae-Sue;Hwang, Ki-Jun;Mar, Woong-Chon
    • Natural Product Sciences
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    • v.2 no.2
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    • pp.130-136
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    • 1996
  • Evaluation of plant extracts that might inhibit hepatitis B virus (HBV) replication was performed to find potent anti-HBV agents. Eighty-five species of plants from forty-three families were tested for their anti-HBV activities using HBV-producing HepG2-derived 2.2.15 cells. The anti-HBV activity of plant extracts was measured by slot blot hybridization technique and cytotoxicity was determined by crystal violet staining procedure. All plants were extracted with methanol and the extracts were partitioned into n-hexane, ethyl acetate and aqueous layer. The ethyl acetate fractions of Rhus verniciflua $(stem:\;EC_{50},\;8.2{\mu}g/ml;\;CC_{50},\;9.4{\mu}g/ml)$, Gastrodia elata $(root:\;EC_{50},\;17.7{\mu}g/ml;\;CC_{50},\;>20{\mu}g/ml)$, Raphanus sativus $(seeds:\;EC_{50},\;17.3{\mu}g/ml;\;CC_{50},\;>20{\mu}g/ml)$, and Angelica gigas $(root:\;EC_{50},\;8.3{\mu}g/ml;\;CC_{50},\;15.6{\mu}g/ml)$ revealed the anti-HBV activity in 2.2.15 cell culture system and these fractions are under the process of further sequential fractionation by column chromatography to find the active principles against HBV.

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Turnip Mosaic Virus Isolated from Rorippa islandica Borb (속속이풀(Rorippa islandica Borb.)에서 분리한 순무 모자이크 바이러스)

  • 최준근;최국선;최장경;유병주;정태성
    • Korean Journal Plant Pathology
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    • v.10 no.2
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    • pp.136-139
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    • 1994
  • Turnip mosaic virus (TuMV) was isolated from Rorippa islandica showing mild mosaic symptom in growing field of Chinese cabbage and radish. Identification of the virus was based on host range, transmission by aphids, electron micrograph, serological reaction and hybridization detection. The virus systemically infected on Chenopodium quinoa, Nicotiana clevelandii, N. glutinosa, Brassica rapa, B. campestris subsp. pekinensis and Raphanus sativus, whereas showed local infection on C. amaranticolor, Gomphrena globosa and Tetragonia tetragonoides. The virus was transmitted by aphid (Myzus persicae). The virus particle was filamentous with 720$\times$12 nm in length, and reacted positively with an antiserum of TuMV in agar gel double duffusion test. In slot-blot hybridization using the digoxigenin(DIG)-labeled RNA probe, TuMV-RNA could be detected in sap of R. islandica infected with the virus. This is the first report of a natural infection of that virus on R. islandica.

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Detection of Plant RNA Viruses by Hybridization Using In Vitro Transcribed RNA Probes (In Viro 전사 RNA Probe를 이용한 식물 바이러스병의 진단)

  • 최장경;이종희;함영일
    • Korean Journal Plant Pathology
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    • v.11 no.4
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    • pp.367-373
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    • 1995
  • The cDNAs derived from the coat protein (CP) genes of six plant RNA viruses, tobacco mosaic virus-pepper strains (TMV-P) and -ordinary strain (TMV-OM), potato virus Y (PVY), turnip mosaic virus (TuMV), cucumber mosaic virus (CMV) and potato leafroll virus (PLRV), were subcloned into the transcription vector, pSPT18, containing SP6 and T7 promoters. The digoxigenin (DIG)-labeled RNA polymerase after linearlization of the cloned pSPTs with XbaI or SacI, and were tested for their sensitivities for the detection of the six viruses. In slot-blot hybridization, dilution end points for the detection of TMV-P and TMV-OM were 10-4, while those of PVY, TuMV and CMV were 10-3. PLRV was detected at the dilution of 10-2. When each RNA probe was applied for the detection of the viruses in the preparations from the leaf disks (8 mm in diameter, and 12 to 15 mg in weight) of infected natural host plants, TMV-P, TMV-OM and TuMV could be detected from one disk, while PVY from 1 or 2 disks. CMV was detected in the preparation from two disks, and PLRV from three disks. With DIG-labeled RNA probe, PVY was detected at 5 days after inoculation, but with ELISA the virus was detected at 8 days after inoculation to tobacco (Nicotiana tabacum cv. Xanthi nc) plants on which symptoms appeared at 9 days after inoculation. No difference was observed in cross reaction between the RNA probes for the detection of TMV-P and TMV-OM.

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Comparative Quantitative Study of Surfactant Protein C mRNA by Filter Hybridization and Solution Hybridization in Rats (Filter Hybridization과 Solution Hybridization 방법에 의한 백서 Surfactant Protein C mRNA 정량측정의 비교)

  • Kim, Jin-Ho;Sohn, Jang-Won;Yang, Seok-Chul;Yoon, Ho-Joo;Shin, Dong-Ho;Park, Sung-Soo
    • Tuberculosis and Respiratory Diseases
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    • v.51 no.6
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    • pp.517-529
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    • 2001
  • Background : Surfactant protein C(SP-C) is a hydrophobic 5,000 dalton molecule. SP-C has the primary roles in accelerating surface spreading of a surfactant phospholipid. The filter hybridization and solution hybridization assays are both rapid and sensitive and can be used to measure the RNAs complementary to any cloned DNA sequence. Methods : The authors measured the SP-C mRNA levels quantitatively using solution hybridization and filter hybridization assays to obtain a standard curve equation to quantify the mRNA of unknown samples comparatively. Results : 1. The minimum level of the specimens by solution hybridization was 3 pg for SP-C mRNA. 2. The standard curve equation of the solution hybridization assay between the counts per minute(Y) and the SP-C mRNA transcript input(X) was Y=6.46 X+244. The correlation coefficient was 0.99. 3. The minimum detection level of specimens by filter hybridization was 0.1 ng for SP-C mRNA. 4. The standard curve equation of the filter hybridization assay between the counts per minute(Y) and SP-C mRNA transcript input(X) is Y=2541.6 X+252.7. The correlation coefficient was 0.99. Conclusions : A comparison of CPM/filter in the linear range allowed an accurate and reproducible estimation of the SP-C mRNA copy number. Filter hybridization and solution hybridization assays are both rapid and sensitive and can be used to measure the RNAs complementary to any cloned DNA sequence. It is ideally suited to situations where accurate quantitation of multiple samples is required.

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Effect of Dietary Capsaicin on Proto-oncogenes Expression in Various in Mice (식이 Capsaicin이 마우스의 주요 장기조직에서의 Proto-oncogenes Expression에 미치는 영향)

  • 김정미;한인섭;김병삼;유리나
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.25 no.6
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    • pp.1024-1030
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    • 1996
  • Capsaicin (8-methyl-N-vanillyl-6-nonenamide: CAP) is a mai or ingredient of hot pepper that has been used as a spicy food additive, preservative, and medicine. In this study, we evaluated the effect of dietary CAP on the selected proto-oncogene(c-jun, c-myc, H-ras, erbB, p53) expressions in various tissues of mice. Male ICR mice were divided into four groups and fed the experimental diets containing CAP at the levels of 0, 5, 20 and 100ppm for four weeks. Steady state RNA levels in various tissues were measured by slot blot hybridization assay. C-jun expression level was enhanced in stomach tissue from mice fed 20ppm CAP and significantly reduced from mice fed 100ppm CAP. The c-jun expression levels were differentially altered in organ-specific manner, Tumor suppressor gene p53 expression level appeared to be slightly increased in the liver from mice fed 20ppm CAP. These results suggested that dietary CAP differentially modulates c-jun and p53 expression in various organs.

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The 16S rDNA Gene Sequencing and Specific Probes Designing for the Identification of Edwardsiella tarda

  • Lee Ju Suk;Choi Jae Young;Sim Doo Saing;Kim Hyeung Rak;Jung Tae Sung;Kim Jae Ho;Oh Myung Joo
    • Fisheries and Aquatic Sciences
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    • v.3 no.1
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    • pp.64-70
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    • 2000
  • DNA probes for the l6S rRNA have been designed for the detection of Edwardsiella tarda. In order to accomplish this purpose, the l6S rRNA gene from E. tarda has been cloned and sequenced. Two highly feasible oligonucleotide probe sites have been determined by the database analysis programs presented by PCGENE and BLAST. These two probes have been evaluated by slot blot hybridization analysis. Hetero- and homo-trimeric templates have been synthesized using these two probe sites. The templates have been further multimerized by PCR to generate between 150 and 300 bp long DIG-11-dUTP labeled probes. Unlike 3' end labeled oligonucleotide probes or templates, multimerized probes showed no cross­hybridization in the given experimental condition. Furthermore, a significant increase in sensitivity has been observed with these probes. This method, we presented here, may be useful for the designing of probes for the detection of other fish pathogenic microorganisms also.

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