• Title/Summary/Keyword: Base pair

Search Result 329, Processing Time 0.021 seconds

Comparison of PCR-RFLP and Real-Time PCR for Allelotyping of Single Nucleotide Polymorphisms of RRM1, a Lung Cancer Suppressor Gene (폐암 억제유전자 RRM1의 단일염기다형성 검사를 위한 PCR-RFLP법과 Real-Time PCR법의 유용성 비교)

  • Jeong, Ju-Yeon;Kim, Mi-Ran;Son, Jun-Gwang;Jung, Jong-Pil;Oh, In-Jae;Kim, Kyu-Sik;Kim, Young-Chul
    • Tuberculosis and Respiratory Diseases
    • /
    • v.62 no.5
    • /
    • pp.406-416
    • /
    • 2007
  • Background: Single nucleotide polymorphisms (SNPs), which consist of a substitution of a single nucleotide pair, are the most abundant form of genetic variations occurring with a frequency of approximately 1 per 1000 base pairs. SNPs by themselves do not cause disease but can predispose humans to disease, modify the extent or severity of the disease or influence the drug response and treatment efficacy. Single nucleotide polymorphisms (SNPs), particularly those within the regulatory regions of the genes often influence the expression levels and can modify the disease. Studies examining the associations between SNP and the disease outcome have provided valuable insight into the disease etiology and potential therapeutic intervention. Traditionally, the genotyping of SNPs has been carried out using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP), which is a low throughput technique not amenable for use in large-scale SNP studies. Recently, TaqMan real-time PCR chemistry was adapted for use in allelic discrimination assays. This study validated the accuracy and utility of real-time PCR technology for SNPs genotyping Methods: The SNPs in promoter sequence (-37 and -524) of lung cancer suppressor gene, RRM1 (ribonucleotide reductase M1 subunit) with the genomic DNA samples of 89 subjects were genotyped using both real-time PCR and PCR-RFLP. Results: The discordance rates were 2.2% (2 mismatches) in -37 and 16.3% (15 mismatches) in -524. Auto-direct sequencing of all the mismatched samples(17 cases) were in accord with the genotypes read by real-time PCR. In addition, 138 genomic DNAs were genotyped using real-time PCR in a duplicate manner (two separated assays). Ninety-eight percent of the samples showed concordance between the two assays. Conclusion: Real-time PCR allelic discrimination assays are amenable to high-throughput genotyping and overcome many of the problematic features associated with PCR-RFLP.

Characterization of Bruton's Tyrosine Kinase Genetic Mutations in One Korean X-linked Agammaglobulinemia Family (반성 열성 범저감마글로불린혈증 1가계 3환자의 Bruton's Tyrosine Kinase 유전자 변이 및 임상 양상)

  • Jo, Eun-Kyeong;Song, Chang-Hwa;Park, Jeong-Kyu;Baek, Young-Jong;Rhu, Hye-Young;Lee, Jae-Ho;Hwang, Tai-Ju;Kook, Hoon
    • Clinical and Experimental Pediatrics
    • /
    • v.45 no.2
    • /
    • pp.183-191
    • /
    • 2002
  • Purpose : X-linked agammaglobulinemia(XLA) is an immunodeficiency caused by abnormalities in Bruton's tyrosine kinase(Btk), and is characterized by a deficiency of peripheral blood B cells. We studied the cytoplasmic expression of Btk protein and analyzed the Btk gene in peripheral blood mononuclear cells from two siblings and one cousin with XLA, as well as additional family members. Methods : Btk protein expression was analyzed by flow cytometry. Isolation of the coding sequence of the Btk gene was performed by amplification using the reverse transcription-polymerase chain reaction(RT-PCR) technique. Sequence alterations were screened by the single-stranded conformation polymorphism(SSCP) method and characterized by standard sequencing protocols. Results : Cytoplasmic expression of Btk protein in monocytes was not detected in three patients with XLA. In addition, Btk protein analysis clearly showed cellular mosaicism in monocytes from four obligate carriers, findings further supported by SSCP. A single base pair mutation(T to C) in Btk-exon three, which encodes the PH domain, was identified in four XLA patients. A diagnostic sequencing analysis was established to detect heterozygotic pattern in 4 carrier females. Furthermore, we found significant clinical heterogeneity in individuals with the same gene mutation. Conclusion : The implicating genetic alteration provided valuable clues to the pathogenesis of XLA in Korea and the flow cytometric analysis was suggested as a useful tool for rapid detection of XLA patients and carriers. The present study has identified a genetic mutation in the Btk coding region and demonstrated heterogeneity in clinical manifestations among patients with the same mutation. A flow cytometric analysis was found to be informative in establishing a deficiency of Btk protein in both patients and carriers and is recommended as a frontline procedure in the molecular diagnosis and work-up of XLA.

The Effects of Experimental Learning Using Small-Scale Chemistry on the Science Learning Achievement of Elementary School Students and Teachers' Perceptions (Small-Scale Chemistry을 적용한 초등학교 과학실험 수업이 과학 학업성취도에 미치는 영향 및 교사의 인식)

  • Lee, Na-Kyeong;Kim, Sung-Kyu
    • Journal of Science Education
    • /
    • v.38 no.2
    • /
    • pp.302-316
    • /
    • 2014
  • The purpose of this study is to devise a Small-Scale Chemistry (SSC) lab program for primary school learners and to examine its effects on science learning achievement. In addition, it will be examined whether the type of learning groups affects the achievement or not. The participants in the current study were 173 6th graders from 6 classes of Y elementary school in Changwon city, Gyeongnam. Three classes(86) were assigned to the experimental group and the other three, the comparative group after checking the pre-homogeneity between the two groups through t-test on the scores of the science mid-term exam. We conducted five experimental sessions on the Acid and Base in the science textbook for the sixth graders. The students of one experimental class worked in pairs and another class worked individually, but the students of the comparative classes were divided into groups of six(one group with pair, another group with individual work in the SSC program, and the other group conducting the traditional experiment with groups of six students). The data were analyzed by t-test and ANOVA. The results showed that experimental learning using individual work in the SSC program compared to traditional experimental learning was effective in improving science learning achievement. also it was indicated that the teachers could reduce their burden of preparing for classes and of school hours when they utilized the SSC laboratory learning program. Teachers could also actively support students' experimental activities in employing the program. Based on the results, we suggest that the development of the SSC laboratory learning program is meaningful in the sense that this program can help elementary schoolers to improve science learning achievements more than the existing traditional experimental methods.

  • PDF

Mutagenicity of Chloropropanols in SOS Chromotest and Ames Test (SOS Chromotest 및 Ames test에서의 Chloropropanol류의 변이원성)

  • Song, Geun-Seoup;Han, Sang-Bae;Uhm, Tae-Boong;Choi, Dong-Seong
    • Korean Journal of Food Science and Technology
    • /
    • v.30 no.6
    • /
    • pp.1464-1469
    • /
    • 1998
  • SOS Chromotest and Ames test were carried out to evaluate the mutagenicity of three chloropropanols. In the SOS Chromotest, 3-monochloro-l,2-propanediol (3-MCPD) and 2,3-dichloro-1-propanol (2,3-DCP) except for 1,3-dichloro-2-propanol (1,3-DCP) induced SOS response in Escherichia coli PQ37 with dose-response relationship and 2,3-DCP was far more genotoxic than 3-MCPD. The genotoxic activities of both compounds, however, were very lower in E. coli PQ35 (PQ37 $uvrA^+)$ as compared to them in E. coli PQ37, whereas much higher in E. coli PQ243 (PQ37 tagA alkA). These results indicate that there are at least two types of DNA lesions caused by these compounds; one is a excision-repairable and the other is 3-methyladenine or any similar lesion which is excision-unrepairable and can induce adaptive response. In Salmonella typhimurium TA100, all the compounds showed strong mutagenicities, establishing the following genotoxic order: 2,3-DCP>3-MCPD>1,3-DCP. But the mutagenic activities were very low in S. typhimurium TA98 and TA97a. These results suggest that the mutation by chloropropanols can be induced by the DNA lesions causing base-pair substitutions. From the result that the mutagenicities of 3-MCPD and 2,3-DCP in S. typhimurium TA1535 were very low as compared to those in S. typhimurium TA100, it was appeared that the mutations by both compounds necessitate error-prone SOS repair.

  • PDF

Analysis of rpoB Gene in Rifampin-Resistant M. Tuberculosis by Direct Sequencing and Line Probe Assay (염기서열결정과 Line Probe 분석법에 의한 Rifampin내성 결핵균의 rpoB 유전자 분석)

  • Lee, Min-Ki;Kim, Yun-Seong;Lee, Hyo-Jin;Cheon, Du-Su;Yun, Sang-Myung;Park, Sam-Seok;Kim, Cheol-Min;Park, Soon-Kew
    • Tuberculosis and Respiratory Diseases
    • /
    • v.44 no.2
    • /
    • pp.251-263
    • /
    • 1997
  • Background : The emergence of multidrug-resistant strains of Mycobacterium tuberculosis presents a significant challange to the treatment and control of tuberculosis, and there is an urgent need to understand the mechanisms by which strains acquire multidrug resistance. Recent advances in molecular methods for the detection of M. tuberculosis genetic targets have approached the sensitivity of culture. Furthermore the prospect of determining resistance in mycobacteria at the nucleic acid level particulary to first-line drugs like rifampin, isoniazid has provided a glimps of the next generation of sensitivity test for M. tuberculosis. Previous studies in RMP resistant M. tuberculosis have shown that mutation in $\beta$subunit of RNA polymerase is main mechanism of resistance. Method : In this study, rpoB gene for the $\beta$subunit of RNA polymerase from M. tuberculosis of 42 cultured samples (32 were RMP resistant and 10 were sensitive cases) were isolated and characterised the mutations. Direct sequencing data were compared with the results of INNO-LiPA Line Probe Assay (LiPA, Innogenetics, Belgium), commercial RMP resistance detecting kit using reverse hybridization method. Results : All of the RMP resistant samples were revealed the presence of mutation by LiPA. In 22 samples (68.8%) out of 32 RMP resistant cases, the mutation types were confirmed by the positive signal at one of 4 mutation bands in the strip. The most frequent type was R5 (S531L) which were 17 cases (77.3%). Results of direct sequencing were identified the exact characteristics of 8 mutations which were not confirmed by LiPA. S522W type point mutation and 9 base pair deletion at codon 513~515 were new identified mutations for the first time. Conclusion : Mutations in rpoB gene is the main mechanism of RMP resistance in M. tuberculosis and LiPA is a very useful diagnostic tool for the early diagnosis of RMP resistance in M. tuberculosis.

  • PDF

The Mechanisms of Resistance to TNF in TNF-Sensitive Cancer Cells Transfected with TNF-$\alpha$ Gene Using Retroviral Vector (암세포에서 Retroviral Vector를 이용한 종양괴사인자 유전자 이입후 획득된 종양괴사인자 내성의 기전)

  • Lee, Hyuk-Pyo;Oh, Yeon-Mok;Yoo, Chul-Gyu;Kim, Young-Whan;Shim, Young-Soo;Han, Sung-Koo
    • Tuberculosis and Respiratory Diseases
    • /
    • v.44 no.3
    • /
    • pp.547-558
    • /
    • 1997
  • Background : Tumor necrosis factor(TNF) has been considered as an important candidate for cancer gene therapy based on its potent anti-tumor activity. However, since the efficiency of current techniques of gene transfer is not satisfactory, the majorities of current protocols is aiming the in vitro gene transfer to cancer cells and re-introducing genetically modified cancer cells to hoot. In previous study, it was shown that TNF-sensitive cancer cells transfected with TNF-$\alpha$ cDNA would become highly resistant to TNF. Understanding the mechanisms of TNF-resistance in TNF-$\alpha$ gene transfected cancer cells would be an important step for improving the efficacy of cancer gene therapy as well as for better understandings of tumor biology. This study was designed to evaluate the role of new protective protein synthesis in the acquired resistance to TNF of TNF-$\alpha$ gene transfected cancer cells. Method : We transfected TNF-$\alpha$ c-DNA to WEHI164, a murine fibrosarcoma cell line, using retroviral vector(pLT12SN(TNF)) and confirm the expression of TNF with PCR, ELISA, MIT assay. Then we determined the TNF resistance of TNF gene transfected cells(WEHI164-TNF) and the changes of TNF sensitivities after treatments with actinomycin D(transcription inhibitor) and cycloheximide ( translation inhibitor). Results : WEHI164 which was sensitive to TNF became resistant to TNF after being transfected with TNF-$\alpha$ gene and the resistance to TNF was partially reversed after treatment with actinomycin D, but not with cycloheximide. Conclusion : The acquired resistance to TNF after TNF-$\alpha$ gene transfection may be associated with synthesis of some protective proteins.

  • PDF

The Role of MnSOD in the Mechanisms of Acquired Resistance to TNF (TNF에 대한 내성획득에서 MnSOD의 역할에 관한 연구)

  • Lee, Hyuk-Pyo;Yoo, Chul-Gyu;Kim, Young-Whan;Han, Sung-Koo;Shim, Young-Soo
    • Tuberculosis and Respiratory Diseases
    • /
    • v.44 no.6
    • /
    • pp.1353-1365
    • /
    • 1997
  • Background : Tumor necrosis factor(TNF) has been considered as an important candidate for cancer gene therapy based on its potent anti-tumor activity. However, since the efficiency of current techniques of gene transfer is not satisfactory, the majority of current protocols is aiming the in vitro gene transfer to cancer cells and re-introducing genetically modified cancer cells to host. In the previous study, it was shown that TNF-sensitive cancer cells transfected with TNF-$\alpha$ cDNA would become highly resistant to TNF, and the probability was shown that the acquired resistance to TNF might be associated with synthesis of some protective protein. Understanding the mechanisms of TNF -resistance in TNF-$\alpha$ cDNA transfected cancer cells would be. an important step for improving the efficacy of cancer gene therapy as well as for better understandings of tumor biology. This study was designed to evaluate the role of MnSOD, an antioxidant enzyme, in the acquired resistance to TNF of TNF-$\alpha$ cDN A transfected cancer cells. Method : We transfected TNF-$\alpha$ c-DNA to WEHI164(murine fibrosarcoma cell line), NCI-H2058(human mesothelioma cell line), A549(human non-small cell lung cancer cell line), ME180(human cervix cancer cell line) cells using retroviral vector(pLT12SN(TNF)) and confirm the expression of TNF with PCR, ELISA, MIT assay. Then we determined the TNF resistance of TNF-$\alpha$ cDNA transfected cells(WEHI164-TNF, NCIH2058-TNF, A549-TNF, ME180-TNF) and the changes of MnSOD mRNA expressions with Northern blot analysis. Results : The MnSOD mRNA expressions of parental cells and genetically modified cells of WEHI164 and ME180 cells(both are naturally TNF sensitive) were not significantly different The MnSOD mRNA expressions of genetically modified cells of NCI-H2058 and A549(both are naturally TNF resistant) were higher than those of the parental cells, while those of parental cells with exogenous TNF were also elevated. Conclusion : The acquired resistance to TNF after TNF-$\alpha$ cDNA transfection may not be associated with the change in the MnSOD expression, but the difference in natural TNF sensitivity of each cell may be associated with the level of the MnSOD expression.

  • PDF

Effect of Retrovirus Mediated TNF-$\alpha$ Gene Transfer to Tumor Necrosis Factor(TNF) Sensitive Tumor Cell Lines on Sensitivity to TNF (Retroviral Vector를 이용한 TNF-$\alpha$ 유전자의 이입이 암세포의 종양괴사인자(TNF) 감수성에 미치는 효과)

  • Oh, Yeon-Mok;Park, Kyeo-Yeong;Jung, Man-Pyo;Yoo, Chul-Gyu;Kim, Young-Whan;Han, Sung-Goo;Sim, Young-Soo;Han, Yong-Chol
    • Tuberculosis and Respiratory Diseases
    • /
    • v.41 no.2
    • /
    • pp.87-96
    • /
    • 1994
  • Background : Since tumor necrosis factor was discovered in 1975, TNF has been well known about its cytotoxic effect on tumor cells in vivo and in vitro. According to the recent improvement of molecular biological techinques, it is possible that exogenous TNF gene is transferred to tumor cells and is expressed in theirs. By virtue of TNF gene transfer, we have expected that TNF expressed in TNF-gene-transferred tumor cells would kill tumor cells in vivo without systemic side effect. The expected mechanisms in which antitumor effects of TNF expressed in TNF-gene-transferred tumor cells are working would be as followings. In the first mechanism, TNF expressed in TNF-gene-transferred tumor cells would kill tumor cells around(like homicide). In the second mechanism, TNF expressed in TNF-gene-transferred tumor cells would kill themselves(like suicide). In the third mechanism, TNF expressed in TNF-gene-transferred tumor cells would recruit immune effector cells and kill tumor cells indirectly. In the last mechanism, TNF expressed in TNF-gene-transferred tumor cells would augment cytokine such as interferon-$\gamma$ to kill tumor cells. Among these four mechanisms of antitumor effect, only the second mechanism has not been established yet. Therefore, to elucidate the second mechanism, We performed this study. Method : We transferred TNF-$\alpha$ gene to NCI-H2058, a human mesothelioma cell line and WEHI164, a murine fibrosarcoma cell line by using retroviral vector(pLT12SNTNF). And, We determined by using MTT assay whether TNF expressed in TNF-gene-transferred tumor cell lines would kill themselves like suicide or not. Then, if TNF-gene-transferred tumor cell lines would not suicide themselves, I would know more about the TNF sensitivity of TNF-gene-transferred tumor cell lines to exogenous TNF also by MTT assay. Result : NCI-H2058 and WEHI164 which were sensitive to TNF, became far less sensitive to endogenous and exogenous TNF after being transferred TNF-$\alpha$ gene to. Conclusion : TNF-gene-transfer to NCI-H2058 and WEHI164 gave them resistance to TNF.

  • PDF

The Effect of Corporate Association on the Perceived Risk of the Product (소비자의 제품 지각 위험에 대한 기업연상과 효과: 지식과 관여의 조절적 역활을 중심으로)

  • Cho, Hyun-Chul;Kang, Suk-Hou;Kim, Jin-Yong
    • Journal of Global Scholars of Marketing Science
    • /
    • v.18 no.4
    • /
    • pp.1-32
    • /
    • 2008
  • Brown and Dacin (1997) have investigated the relationship between corporate associations and product evaluations. Their study focused on the effects of associations with a company's corporate ability (CA) and its corporate social responsibility (CSR) on consumers' product evaluations. Their study has found that both of CA and CSR influenced product evaluation but CA association has a stronger effect than CSR associations. Brown and Dacin (1997) have, however, claimed that there are few researches on how corporate association impacts product responses. Accordingly, some of researchers have found the variables to moderate or to mediate the relationship between the corporate association and the product responses. In particular, there has been existed a few of studies that tested the influence of the reputation on the product-relevant perceived risk, but the effects of two types of the corporate association on the product-relevant perceived risk were not identified so far. The primary goal of this article is to identify and empirically examine some variables to moderate the effects of CA association and CSR association on the perceived risk of the product. In this articles, we take the concept of the corporate associations that Brown and Dacin (1997) had proposed. CA association is those association related to the company's expertise in producing and delivering its outputs and CSR association reflected the organization's status and activities with respect to its perceived societal obligations. Also, this study defines the risk, which is the uncertainty or loss of the product and corporate that consumers have taken in a particular purchase decision or after having purchased. The risk is classified into product-relevant performance risk and financial risk. Performance risk is the possibility or the consequence of a product not functioning at some expected level and financial risk is the monetary loss one perceives to be incurring if a product does not function at some expected level. In relation to consumer's knowledge, expert consumers have much of the experiences or knowledge of the product in consumer position and novice consumers does not. The model tested in this article are shown in Figure 1. The model indicates that both of CA association and CSR association influence on performance risk and financial risk. In addition, the effects of CA and CSR are moderated by product category knowledge (product knowledge) and product category involvement (product involvement). In this study, the relationships between the corporate association and product-relevant perceived risk are hypothesized as the following form. For example, Hypothesis 1a($H_{1a}$) is represented that CA association has a positive influence on the performance risk of consumer. Also, the hypotheses that identified some variables to moderate the effects of two types of corporate association on the perceived risk of the product are laid down. One of the hypotheses of the interaction effect is Hypothesis 3a($H_{3a}$), it is described that consumer's knowledges of the product moderates the negative relationship between CA association and product-relevant performance risk. A field experiment was conducted in order to examine our model. The company tested was not real but imagined to meet the internal validity. Water purifiers were used for our study. Four scenarios have been developed and described as the imaginary company: Type A with both of superior CA and CSR, Type B with superior CSR and inferior CA, Type C with superior CA and inferior CSR, and Type D with both inferior of CA and CSR. The respondents of this study were classified into four groups. One type of four scenarios (Type A, B, C, or D) in its questionnaire was given to the respondent who filled out questions. Data were collected by means of a self-administered questionnaire to the respondents, chosen in convenience. A total of 300 respondents filled out the questionnaire but 207 were used for further analysis. Table 1 indicates that the scales in this study are reliable because the range of coefficients of Cronbach's $\alpha$ are from 0.85 to 0.92. The composite reliability is in the range of 0,85 to 0,92 and average variance extracted is in 0.72-0.98 range that is higher than the base level of 0.6. As shown in Table 2, the values for CFI, NNFI, root-mean-square error approximation (RMSEA), and standardized root-mean-square residual (SRMR) are acceptably close to the standards suggested by Hu and Bentler (1999):.95 for CFI and NNFI,.06 for RMSEA, and.08 for SRMR. We also tested discriminant validity provided by Fornell and Larcker (1981). As shown in Table 2, we found strong evidence for discriminant validity between each possible pair of latent constructs in all samples. Given that these batteries of overall goodness-of-fit indices were accurate and that the model was developed on theoretical bases, and given the high level of consistency across samples, this enables us to proceed the previously defined scales. We used the moderated hierarchical regression analysis to test the influence of the corporate association(CA and CSR associations) on product-relevant perceived risk(performance and financial risks) and to identify the variables moderating the relationship between the corporate association and product-relevant performance risk. In this study, dependent variables are performance and financial risk. CA and CSR associations are described the independent variables. The moderating variables are product category knowledge and product category involvement. The results are, as expected, found that CA association has statistically a significant influence on the perceived risk of the product, but CSR association does not. Product category knowledge and involvement moderate the relationship between the CA association and the perceived risk of the product. However, the effect of CSR association on the perceived risk of the product is not moderated by the consumers' knowledge and involvement. For this result, it is necessary for a corporate to inform its customers CA association more than CSR association so that they could be felt to be the reduction of the perceived risk. The important theoretical contribution of this research is the meanings that two types of corporate association that Brown and Dacin(1997), and Brown(1998) have proposed replicated the difference of the effects on product evaluation. According to Hunter(2001), it was an important affair to accomplish the validity of a particular study and we had to take about ten studies to deduce a strict study. Next, there is the contribution of the this study to find that the effects of corporate association on the perceived risk of the product are varied by the moderator variables. In particular, the moderating effect of knowledge on the relationship between corporate association and product-relevant perceived risk has not been tested in Korea. In the managerial implications of this research, we suggest the necessity to stress the ability that corporate manufactures the product well(CA association) than the accomplishment of corporate's social obligation(CSR association). This study suffers from various limitations that imply future research directions. The moderating effects of product category knowledge and involvement on the relationship between corporate association and perceived risk need to be replicated. Next, future research could explore whether the mediated effects of the perceived risk has the relationship between corporate association and consumer's product purchase. In addition, to ensure the external validity of the study will be needed to use realistic company, not artificial.

  • PDF