• Title/Summary/Keyword: Filter hybridization

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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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Quantitative Measurement of Surfactant Protein B mRNA by Filter Hybridization (Filter Hybridization 방법에 의한 Surfactant Protein B mRNA의 정량측정)

  • Park, Sung-Soo;Lee, Dong-Hoo;Shin, Dong-Ho;Lee, Jung-Hee
    • Tuberculosis and Respiratory Diseases
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    • v.39 no.3
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    • pp.242-247
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    • 1992
  • Background: The ability to precisely measure specific mRNA levels by hybridization to complementary DNA probes is an important tool for analyzing the regulation of gene expression. Surfactant proteins have important roles in regulating surfactant metabolism as well as in determing its physical properties. Method: The complete coding regions for rat surfactant protein complementary DNA of surfactant protein B were subcloned into pGem 3Z or 4Z such that either antisense or sense transcripts were obtained by using SP 6 RNA polymerase. Surfactant protein B mRNA was measured by filter hybridization. Results: Equation of standard curve between counts per minute (Y) and surfactant protein B mRNA transcript input (X) was Y=2034.9 X+159.1. Correlation coefficient was 1.0. Couclusions: Filter hybridization assay is suited to situation when rapid, accurate quantitation of multiple samples is required.

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Assessment of the Specificity of A Hybridization of Surfactant Protein A by Addition of Non-specific Rat Spleen RNA (Surfactant Protein A mRNA을 이용한 유전자 재결합 반응에서 비특이성 RNA의 첨가에 의한 특이성 검정)

  • Kim, Byeong Cheol;Kim, Mi Ok;Kim, Tae-Hyung;Sohn, Jang Won;Yoon, Ho Joo;Shin, Dong Ho;Park, Sung Soo
    • Tuberculosis and Respiratory Diseases
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    • v.56 no.4
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    • pp.393-404
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    • 2004
  • Background : Nucleic acid hybridization has become an essential technique in the development of our understanding of gene structure and function. The quantitative analysis of hybridization has been used in the measurement of genome complexity and gene copy number. The filter hybridization assay is rapid, sensitive and can be used to measure RNAs complementary to any cloned DNA sequence. Methods : The authors assessed the accuracy, linearity, correlation coefficient and specificity of the hybridization depending on the added dose(0, 1, 5, and $10{\mu}g$) of non-specific rat spleen RNA to hybridization of surfactant protein A mRNA. Filter hybridization assays were used to obtain the equation of standard curve and thereby to quantitate the mRNA quantitation. Results : 1. Standard curve equation of filter hybridization assay between counts per minute (X) and spleen RNA input (Y) was Y=0.13X-19.35. Correlation coefficient was 0.98. 2. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) was Y=0.00066X-0.046. Correlation coefficient was 0.99. 3. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of $1{\mu}g$ spleen RNA was Y=0.00056X-0.051. Correlation coefficient was 0.99. 4. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of $5{\mu}g$ spleen RNA was Y=0.00065X-0.088. Correlation coefficient was 0.99. 5. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of $10{\mu}g$ spleen RNA was Y=0.00051X-0.10. Correlation coefficient was 0.99. Conclusions : Comparison of cpm/filter in a linear range allowed accurate and reproducible estimation of surfactant protein A mRNA copy number irrespective of the addition dosage of non-specific rat spleen RNA over the range $0-10{\mu}g$.

Community structure analysis of nitrifying biofilms by 16S rRNA targeted probe and fluorescence in situ hybridization (FISH)

  • Han, Dong-U;Kim, Dong-Jin
    • 한국생물공학회:학술대회논문집
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    • 2001.11a
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    • pp.282-285
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    • 2001
  • The microbial community structure and in situ spatial distribution of ammonia oxidizing and nitrite oxidizing bacteria in nitrifying biofilm of an upflow biological aerated filter system were investigated. The reactor had been continuously operated under high free ammonia concentration and low DO concentration for nitrite accumulation more than 2 years before the experiment. Fluorescence in situ hybridization

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Nucleotide Sequence Homology in Rotaviruses (Rotaviruses의 염기배열 유사성 측정)

  • ;Spendlove, Rex S.;Barnett, Bill B
    • Korean Journal of Microbiology
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    • v.26 no.3
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    • pp.155-161
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    • 1988
  • Nucleotide sequence homology between bovine, simian, and porcine rotavirus was determined by the RNA:RNA hybridization technique. Single stranded RNA, prepared in vitro with EDTA activated endogeneous viral RNA polymerase, was hhbridized with tritium labeled bovine rotavirus genomic RNA. The heteroduplex RNA was treated with single stranded RNA specific ribonucleases and the RNase resistant hybrid RNA was precipitated, and collected by filtration on a filter paper. Seventy four percent RNA sequence homology between bovine and simian rotavirus and 8 percent RNA sequence homology between bovine and porcine rotavirus was confirmed by hybridization between tritium labeled single stranded RNA and viral genomic RNA.

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Detection of Waterborne Pathogens in Public Bath Houses by PCR-Reverse Blot Hybridization Assay (PCR-REBA) (분자생물학적 방법인 PCR-REBA를 이용한 대중목욕탕 수질 중 수인성병원성미생물 검출)

  • Song, Woon-Heung;Choi, Seung-Gu;Yang, Byoung-Seon;Lee, Jae-Sang
    • Journal of the Korea Academia-Industrial cooperation Society
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    • v.12 no.8
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    • pp.3517-3522
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    • 2011
  • Contamination of public bath water by waterborne pathogens can cause disease outbreaks and contribute to background rates of disease. The aim of this study is to determine the prevalence of waterborne pathogens in public baths. A total of 30 water samples were collected from 30 different public baths in seoul, Korea. Pathogens in water samples were concentrated by 0.45 ${\mu}m$ nitrocellulose membrane filter, analyzed by both cultivation and polymerase chain reaction-reverse blot hybridization (PCR-REBA) of partial 16S rRNA gene. Various microorganisms including Escherichia coli and Shigella spp. were identified by microbiological cultivation. E. coli, Shigella spp., Salmonella spp., Pseudomonas spp. and Mycobacterium spp. were identified by PCR-REBA. Our results suggest that appropriate hygiene practice and continuous monitoring is needed for reducing health risk associated with public bath houses.

Analysis on the distribution of nitrogen and phosphorus removing microorganisms and nitrifying activity in a trickling filter (살수여상에서의 질소, 인 제거 미생물 분포 및 질산화 활성 조사)

  • Kim, Dong-Jin;Yoo, Ik-Keun;Ahn, Dae-Hee
    • Journal of Environmental Science International
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    • v.18 no.6
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    • pp.691-698
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    • 2009
  • Trickling filter has been extensively studied for the domestic wastewater treatment especially for the small scale plants in rural area. The performance of the trickling filter depends on the microbial community and their activity in the biofilms on the media. Nitrification. denitrification, and phosphorus removal of the trickling filter from the wastewater depend on the activity and the amount of the specific microorganisms responsible for the metabolism. For the estimation of the performance of a trickling filter, batch nitrification experiment and fluorescence in situ hybridization (FISH) were carried out to measure the microbial activity and its distribution on the media of the trickling filter. Batch nitrification activity measurement showed that the top part of the 1st stage trickling filter had the highest nitrification activity and the maximum activity was 0.002 g $NH_4$-N/g MLVSS${\cdot}$h. It is thought that higher substrate (ammonia) concentration yields more nitrifying bacteria in the biofilms. The dominant ammonia oxidizer and nitrite oxidizer in the biofilm were Nitrosomonas species and genus Nitrospira, respectively, by FISH analysis. Less denitrifiers were found than nitrifiers in the biofilm by the probe Rrp1088 which specifically binds to Rhodobacter, Rhodovulum, Roseobacter, and Paracoccus. Phosphorus accumulating bacteria were mostly found at the surface of the biofilm by probe Rc988 and PAO651 which specifically binds to Rhodocyclus group and their biomass was less than that of nitrifiers.

Molecular Cloning and Alternative Splicing of Growth Hormone Transcripts in Greenling, Hexagrammos otakii (쥐노래미 (Hexagrammos otakii) 성장호르몬 cDNA유전자의 염기서열 변이 및 발현 특성)

  • Nam Yoon Kwon;Kim Dong Soo
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.35 no.6
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    • pp.676-681
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    • 2002
  • Different types of transcripts encoding growth hormone (GH) were identified from cDNA libraries constructed with pituitaries of a marine fish species, greenling (Hexagrammos otakii). GH-homologous cDNA clones were isolated using the high-density filter hybridization and the expressed sequence tag techniques. Of 39 full-length positive cDNA clones, 31 clones ($79\%$) displayed an identical sequence, however, remaining 8 clones exhibited several polymorphisms in their sequences including (1) the length and sequence variability in the 5' upstream region, (2) insertional sequences in open reading frame, and (3) deletion and/or single nucleotide polymorphism in the untranslated 3' region. Based on RT-PCT and RNA dot blot analyses, these transcripts were proven to be expressed in a pituitary-specific manner.

Detection of Campylobacter jejuni in food and poultry visors using immunomagnetic separation and microtitre hybridization

  • Simard, Ronald-E.
    • Proceedings of the Korean Society of Fisheries Technology Conference
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    • 2000.05a
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    • pp.71-73
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    • 2000
  • Campylobacter jejuni is most frequently identified cause of cause of acute diarrhoeal infections in developeed countries, exceeding rates of illness caused by both salmonella and shigilla(Skirrow, 1990 ; Lior 1994). Previous studies on campylobacter jejuni contamination of commercial broiler carcasses in u.s.(Stern, 1992). Most cases of the disease result from indirect transmission of Campylobactor from animals via milk, water and meat. In addition to Campylobactor jejuni. the closely relates species Campylobactor coli and Campylobactor lari have also been implicated as agents of gastroenteritis in humans. Campylobactor coli represented only approximately 3% of the Campylobactor isolates from patients with Campylobactor enteritis(Griffiths and Park, 1990) whereas Campylobactor coli is mainly isolated from pork(Lmmerding et al., 1988). Campylobactor jejuni has also been isolated from cases of bacteremia, appendicitis and, recently, has been associated with Guillai-Barre syndrome(Allos and Blaser, 1994; von Wulffen et al., 1994; Phillips, 1995). Studies in volunteers indicated that the infectious dose for Campylobactor jejuni is low(about 500 organisms)(Robinson, 1981). The methods traditionally used to detect Campylobactor ssp. in food require at least two days of incubation in an enrichment broth followed by plating and two days of incubation on complex culture media containing many antibiotics(Goossens and Butzler, 1992). Finnaly, several biochemical tests must be done to confirm the indentification at the species level. Therfore, sensitive and specific methods for the detection of small numbers of Campylobactor cells in food are needed. Polymerase chain reaction(PCR) assays targeting specific DNA sequences have been developed for the detection of Campylobactor(Giesendorf and Quint, 1995; Hemandex et al., 1995; Winter and Slavidk, 1995). In most cases, a short enrichment step is needed to enhance the sensitivity of the assay prior to detection by PCR as the number of bacteria in the food products is low in comparison with those found in dinical samples, and because the complex composition of food matrices can hinder the PCR and lower its sensitivity. However, these PCR systems are technically demanding to carry out and cumbersome when processing a large number of samples simutaneously. In this paper, an immunomagnetic method to concentrate Campylobactor cells present in food or clinical samples after an enrichment step is described. To detect specifically the thermophilic Campylobactor. a monoclonal antibody was adsorbed on the surface of the magnetic beads which react against a major porin of 45kDa present on the surface of the cells(Huyer et al., 1986). After this partial purification and concentration step, detection of bound cells was achieved using a simple, inexpensive microtitre plate-based hybridization system. We examined two alternative detection systems, one specific for thermophilic Campylobactor based on the detection of 23S rRNA using an immobilized DNA probe. The second system is less specific but more sensitive because of the high copy number of the rRNA present in bacterial cell($10^3-10^4$). By using specific immunomagnetic beads against thermophilic Campylobactor, it was possible to concentrate these cells from a heterogeneous media and obtain highly specific hybridization reactions with good sensitivity. There are several advantages in using microtitre plates instead of filter membranes or other matrices for hybridization techniques. Microtitre plates are much easier to handle than filter membranes during the adsorption, washing, hybridization and detection steps, and their use faciilitates the simultanuous analysis of multiple sample. Here we report on the use of a very simple detection procedure based on a monoclonal anti-RNA-DNA hybrid antibody(Fliss et al., 1999) for detection of the RNA-DNA hybrids formed in the wells.

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Inhibitory Effects of a Korean Strain Gpf01 Identified as Pseudomonas fluorescens on Cucumber mosaic virus

  • Ipper, Nagesh S.;Kim, Jung-Eun;Koo, Jun-Hak;Hur, Jang-Hyun;Lim, Chun-Keun
    • The Plant Pathology Journal
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    • v.21 no.3
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    • pp.262-269
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    • 2005
  • An antiviral producing bacterial strain was isolated from a ginseng rhizosphere in Kangwon province of Republic of Korea. In order to identify the bacterial strain, microbiological, physiological and biochemical tests were performed, along with RAPD, 16S rRNA, 16S-23S rRNA ITS (intergenic spacer region) and DNA-DNA hybridization analyses. The bacterium was found to be a strain of Pseudomonas fluorescens, which was designated as Gpf01. The strain was grown in Muller-Hinton (MH) broth, and the culture supernatant obtained was filtered through a $0.45{\mu}l$ filter. It was further boiled at $100^{\circ}C$ and tested in two experiments for its ability to control a yellow strain of Cucumber mosaic virus (CMV-Y). In the first experiment, boiled culture filtrate (RCF) was treated on one half of the leaves of Chenopodium amaranticolor followed by CMV- Y inoculation on both halves. In the second experiment, BCF was treated on the lower leaves of Nicotiana tobacum var. Xanthi-nc, with the CMV-Y mechanically inoculated onto the upper untreated leaves. In the first experiment, BCF treatment was able to considerably reduce the number of viral lesion, and in the second experiment, plants treated with BCF showed no visible viral symptoms compared to the Muller-Hinton (MH) media treated controls 15 days post inoculation (dpi), and remained symptomless throughout the study period. Thus, Gpf01, identified as P. fluorescence, was able to produce an antiviral component in the culture filtrate, which was found to be heat stable, non-phytotoxic and effective in local as well as systemic hosts of CMV.