• Title/Summary/Keyword: Oligonucleotide

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Development of DNA Hybridization Detection Sensors and Analysis of Characteristics Using Electrochemical methods (전기화학법을 이용한 DNA Hybridization 검출 센서의 개발 및 특성 해석)

  • Ock, Jin-Young;Kim, Do-Kyun;Chang, Jeong-Soo;Kwon, Young-Soo
    • Proceedings of the KIEE Conference
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    • 2002.11a
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    • pp.260-262
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    • 2002
  • The determination of DNA hybridization can apply the molecular biology research. clinic diagnostics. bioengineering, environment monitoring, food science and other application area. So, The determination of hybridization is very important for the improvement of DNA detection system. In this study, we report the characterization of the DNA hybridization by the electricalchemical methods. The probe oligonucleotide was used to determine the amount of target oligonucleotide in solution using Methylen Blue(MB) as the electrochemical indicators. The cathodic peak currents($I_{peak}$) of MB were linearly related to the concentration of the target oligonucleotide sequence in the range $1[{\mu}M]{\sim}0.1[{\mu}M]$. The detection limit of this approach was 0.01[nM]. As a result, the match oligonucleotide(CR-1) was most stable state and the peak of redox current measured by DNA hybridization detection sensors by using electrochemical method seem to be similar to 1-mer terminal mismatch oligonucleotide(MR-3). The MR-2, MR-3, MR-22 and MR-33 have each mismatching sequence of central and terminal. With this set the role of point mutations was to be investigated. Terminal mismatch oligonucleotide (MR-3, 33) is shown more stable state than central mismatch oligonucleotide(MR-2, 22). And 1-mer mismatch oligonucleotide(MR-2 or 3) is shown more stable state than 2-mer mismatch oligonucleotide(MR-22 or 33).

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Oligonucleotide Chip Analysis of Cervi parvum cornu Herbal-acupuncture Solution (CPC-HAS) on SNU484 carcinomar cells (녹용약침액(鹿茸藥鍼液)의 위암세포주(胃癌細胞柱)에 대한 Oligonucleotide Chip 분석)

  • Ryu, Sung-Hyun;Lee, Kyung-Min;Lee, Bong-Hyo;Jung, Tae-Young;Seo, Jung-Chul;Lim, Seong-Chul
    • Korean Journal of Acupuncture
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    • v.23 no.2
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    • pp.125-136
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    • 2006
  • Objectives: It has long been known about the osteogenic effect of CPC-HAS(cervi parvum cornu herbal-acupuncture solution) on bone tissues. However, it has not been determined the effect of CPC-HAS on cancer cells. The purpose of this study is to screen the CPC-HAS mediated differentially expressed genes..in cancer cells such as SNU484 gastric cancer cell lines. Oligonucleotide microarray approache was employed to screen the differential expression genes. Methods: CPC-HAS was prepared by boiling and stored at $-70^{\circ}C$ until use. Cells were treated with various concentrations of CPC-HAS (0.1, 0.5, 1.5, 10, 20 mg/ml) for 24 h. Cell toxicity was tested by MTT assay. To screen the differentially expressed genes in cancer cells, cells were treated with 1.5 mg/ml of CPC-HAS. For oligonucleotide microarray assay, total RNA was used for gene expression analysis using oligonucleotide Genechip(Human genome U133 Plus 2.0., Affimatrix Co.). Results: It has no cytotoxic effects on SNU484 cell in all concentrations(0.l, 0.5, 1.5, 10, 20 mg/ml). In oligonucleotide microarray assay, in SNU484 cells, the number of more than twofold up-regulated genes was 5 while, the number of more than twofold down-regulated genes was 10. Conclusions: This study showed the screening of CPC-HAS mediated differentially regulated genes using combined approaches of oligonucleotide microarray. The screened genes will be used for the better understanding of the therapeutic effects of CPC-HAS on cancer fields.

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Detection of rpoB Gene Mutation in Rifampin-Resistant M. Tuberculosis by Oligonucleotide Chip (Oligonucleotide chip을 이용한 Rifampin 내성 결핵균의 rpoB 유전자 돌연변이 검출)

  • Park, Soon-Kew;Lee, Min-Ki;Chung, Byung-Seon;Kim, Cheol-Min;Chang, Chul-Hun L.;Park, Hee-Kyung;Jang, Hyun-Jung;Park, Seung-Kyu;Song, Sun-Dae
    • Tuberculosis and Respiratory Diseases
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    • v.49 no.5
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    • pp.546-557
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    • 2000
  • Background : Oligonucleotide chip technology has proven to be a very useful tool in the rapid diagnosis of infectious disease. Rifampin resistance is considered as a useful marker of multidrug-resistance in tuberculosis. Mutations in the rpoB gene coding $\beta$ subunit of RNA polymerase represent the main mechanism of rifampin resistance. The purpose of this study was to develop a diagnosis kit using oligonucleotide chip for the rapid and accurate detection of rifampin-resistance in Mycobacterium tuberculosis. Method : The sequence specific probes for mutations in the rpoB gene were designed and spotted onto the glass slide, oligonucleotide chip. 38 clinical isolates of Mycobacterium were tested. A part of rpoB was amplified, labelled, and hybridized on the oligonucleotide chip with probes. Results were analyzed with a laser scanner. Direct sequencing was done to verify the results. Result : The low-density oligonucleotide chip design어 to determine the specific mutations in the rpoB gene of M. tuberculosis accurately detected rifampin resistance associated with mutations in 28 clinical isolates. Mutations at codons 531, 526, and 513 were confirmed by direct sequencing analysis. Conclusion : Mutant detection using oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of multidrug-resistance in M. tuberculosis.

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Oligonucleotide Array-based Detection and Genotyping of Mollicutes (Acholeplasma, Mycoplasma, and Ureaplasma)

  • Jang, Hyun-Jung;Kim, Hyo-Myeung;Kang, Byeong-Chul;Kim, Cheol-Min;Park, Hee-Kyung
    • Journal of Microbiology and Biotechnology
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    • v.19 no.3
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    • pp.265-270
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    • 2009
  • An oligonucleotide array was developed to detect and genotype mollicutes based on the internal transcribed spacer (ITS) sequence. The results of the assay were compared with those of a PCR-RFLP assay. The proposed oligonucleotide array containing 5 genus- and 23 species-specific probes was able to detect Mycoplasma species, including M. penetrans and M. spermatophilum, that were not detected by the PCR-RFLP assay. Therefore, the results demonstrated that the proposed oligonucleotide array was effective for the detection and discrimination of 23 species, including an acholeplasma, 21 mycoplasmas, and a ureaplasma, and showed promise as a countermeasure to ensure that biological products are safe and of good quality.

Detection of DNA Hybridization Characteristics Using Electrochemical methods (전기화학법을 이용한 DNA Hybridization 특성 검출)

  • Kim, Do-Kyun;Chang, Jeong-Soo;Kwon, Young-Soo
    • Proceedings of the KIEE Conference
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    • 2002.07c
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    • pp.1569-1571
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    • 2002
  • The determination of DNA hybridization can apply the molecular biology research, clinic diagnostics, bioengineering, environment monitoring, food science and other application area. So, The determination of hybridization is very important for the improvement of DNA detection system. In this study, we report the characterization of the DNA hybridization by the electricalchemical methods. A new electrochemical biosensor is described for voltammetric detection of gene sequence related to probe oligonucleotide of bacterium Escherichia coli O157:H7. The biosensor involves the immobilization of a 18-mer probe oligonucleotide, which is complemetary to a specific gene sequence related to Escherichia coli O157:H7 on a gold electrode through specific adsorption. The probe oligonucleotide was used to determine the amount of target oligonucleotide in solution using mitoxantrone(MTX) as the electrochemical indicators. The cathodic peak currents $(I_{peak})$ of MTX were linearly related to the concentration of the target oligonucleotide sequence in the range $1[{\mu}M]{\sim}0.1[nM]$. The detection limit of this approach was 0.01[nM]. In addition, these indicators were capable of selectivity discriminating against various mismatching condition.

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Preparation of Oligonucleotide Arrays with High-Density DNA Deposition and High Hybridization Efficiency

  • Park, Jeong-Won;Jung, Yong-Won;Jung, Young-Hwan;Seo, Jeong-Sun;Lee, Young-Hoon
    • Bulletin of the Korean Chemical Society
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    • v.25 no.11
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    • pp.1667-1670
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    • 2004
  • In DNA microarray produced by DNA-deposition technology, DNA-immobilization and -hybridization yields on a solid support are most important factors for its accuracy and sensitivity. We have developed a dendrimeric support using silylated aldehyde slides and polyamidoamine (PAMAM) dendrimers. An oligonucleotide array was prepared through a crosslinking between the dendrimeric support and an oligonucleotide. Both DNAimmobilization and -hybridization yields on the solid support increased by the modification with the dendrimers. The increase of the immobilization and hybridization efficiency seems to result from a threedimensional arrangement of the attached oligonucleotide. Therefore, our dendrimeric support may provide a simple and efficient solution to the preparation of DNA microarrays with high-density DNA-deposition and high hybridization efficiency.

어류 rhabdovirus의 신속 진단을 위한 oligonucleotide chip의 개발 1. Oligonucleotide chip의 probe 설계와 특이성 확인

  • 김영주;강지희;김수미;박수일;이명숙
    • Proceedings of the Korean Society of Fisheries Technology Conference
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    • 2003.05a
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    • pp.309-310
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    • 2003
  • 현재 어류 질병 바이러스를 검출해내기 위해 이용되는 PCR법이나 ELIZA법 만으로는 신속성을 필요로 하는 바이러스 검출에서는 불리하다(Bruchhof et. al., 1995). 따라서 기존의 유전자 진단법을 대체할 수 있고, 동시에 수백 개 이상의 유전자를 빠른 시간 내에 검색할 수 있는 DNA chip 기술을 이용하여 넙치, 돔등의 해산어와 일부 연어과 어류에 질병을 일으키는 rhabdovirus를 신속하고 정확하게 진단할 수 있는 저밀도 oligonucleotide chip을 개발하고자 한다(Chizhikov et at., 2001). (중략)

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어류 rhabdovirus의 신속 진단을 위한 Oligonucleotide chip의 개발 2. Oligonucleotide chip을 위한 기반 조건 구축

  • 김영주;강지희;임재성;김상봉;이명숙
    • Proceedings of the Korean Society of Fisheries Technology Conference
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    • 2003.05a
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    • pp.311-312
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    • 2003
  • Microarray를 해석하는데 있어서 방해가 되는 요인에는 여러 가지가 있을 수 있다. 예를 들면 probe DNA의 농도, spotting 시의 습도, spot의 크기 및 모양, slide blocking 과정, lab리ing technique, hybri야zation 온도와 시간 그리고 background signal 등이 그것이다(Hegde et at., 2000). 본 연구에서는 전보에서 선정된 rhabdovirus의 96개 probe로 실제 oligonucleotide chip을 만들어 spot의 모양이나 크기에 관계되는 spotting 조건을 확립하고, slide blocking과 slide washing에 따른 background signal을 낮추기 위한 과정을 도입하였다. (중략)

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Cholesterol conjugated spermine as a delivery modality of antisense oligonucleotide

  • Im, Yoon Kyung;Kim, Myung Su;Yoo, Hoon
    • International Journal of Oral Biology
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    • v.38 no.4
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    • pp.155-160
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    • 2013
  • The major issue in the development of nucleic acid based therapeutics is the inefficient delivery of these agents into cells. We prepared cholesterol conjugated spermine and evaluated its usefulness as a delivery modality for antisense oligonucleotides in HeLa-Luc cells. A 2'-O-methyl antisense oligonucleotide sequence, designed to correct splicing at an aberrant intron inserted into a normal luciferase reporter gene, was used for complex formation with cholesterol conjugated spermine. Effective delivery of this antisense agent into nucleus would results in the expression of a luciferasereporter gene product. The cholesterol-spermine formed stable complexes with the antisense oligonucleotide and showed modest delivery activity. Furthermore, this delivery activity was maintained even in the presence of serum proteins, mimicking in vivo conditions. Cholesterol-spermine thus has potential as a delivery system for antisense oligonucleotides into cells.

Effect of Carthami Tinctorii Fructus Herbal-acupuncture Solution(CTF-HAS) on Gene Expression in SNU484 carcinomar cells (Oligonucleotide chip을 이용한 홍화자약침액(紅花子藥鍼液)이 위암세포주(胃癌細胞柱)의 유전자(遺傳子) 발현(發顯)에 미치는 영향(影響))

  • Lee, Kyung-Min;Lim, Seong-Chul;Jung, Tae-Young;Seo, Jung-Chul;Han, Sang-Won
    • Journal of Pharmacopuncture
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    • v.8 no.1
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    • pp.31-40
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    • 2005
  • Objectives : It has long been known about the osteogenic effect of CTF-HAS on bone tissues. However, it has not been determined the effect of CTF-HAS on cancer cells. The purpose of this study is to screen the CTF-HAS mediated differentially expressed genes in cancer cells such as SNU484 gastric cancer cell lines. Oligonucleotide microarray approach were employed to screen the differential expression genes. Methods : CTF-HAS was prepared by boiling and stored at $-70^{\circ}C$ until use. Cells were treated with various concentrations of CTF-HAS(0.1, 0.5, 1.5, 10, 20mg/ml) for 24 h. Cytotoxicity was tested by MTT assay. To screen the differentially expressed genes in cancer cells, cells were treated with 1.5mg/ml of CTF-HAS. For oligonucleotide microarry assay, total RNA was used for gene expression analysis using oligonucleotide genechip (Human genome U133 Plus 2.0., Affimatrix Co.). Results : It has no cytotoxic effects on HepG2 cells in all concentration (0.1, 0.5, 1.5, 10,20mg/ml). More than twofold up-regulated genes were 5 genes. The number of more than twofold down-regulated genes was 10. Discussion : This study showed the screening of CTF-HAS mediated differentially regulated genes using combined approaches of oligonucleotide microarray. The screened genes will be used for the better understanding in therapeutic effect of CTF-HAS on cancer field.