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MEC Seminar Proteome Research Lab 2003. 5. 20 Summarized by Park, Ji-Yoon

Integrated Microsystem of Isothermal Amplification of DNA and Electrophoresis on a Microfabricated Plastic Chip for Detection of Specific Gene and Analysis of Genetic Materials. Proceedings of the uTAS 2002 Symposium , Vol. 1, pp 215-217. MEC Seminar Proteome Research Lab 2003. 5. 20

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MEC Seminar Proteome Research Lab 2003. 5. 20 Summarized by Park, Ji-Yoon

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  1. Integrated Microsystem of Isothermal Amplification of DNA and Electrophoresis on a Microfabricated Plastic Chip for Detection of Specific Gene and Analysis of Genetic Materials Proceedings of the uTAS 2002 Symposium, Vol. 1, pp 215-217 MEC Seminar Proteome Research Lab 2003. 5. 20 Summarized by Park, Ji-Yoon

  2. Continuous-Flow PCR on ChipScience, 280, 1046-1048(1998) Fig 1. Schematic of a chip for flow through PCR 7

  3. A Miniature Integrated Device for Automated Multiple Genetic AssaysNucleic Acids Research, 28, e60 (2000) Fig 4. An integrated device that automatically performs a multistep HIV genotyping assay 8

  4. High Sensitivity PCR Assay in Plastic Micro Reactors Lab on a Chip, 2(4), 179-187(2002) 9

  5. Infrared-mediated Thermocycling for DNA Amplification and Electrophoretic Separation on an Integrated Microrchip DeviceProceedings of the uTAS 2002 Symposium, Vol. 1, pp 193-194 Fig 2. Microchip and thermocycling profile 10

  6. Anal. Chem. 74, 3063-3070 (2002) Fig 3. Monolithic integrated polycarbonated DNA assay device DNA Amplification and Hybridization Assays in Integrated Plastic Monolithic DevicesProceedings of the uTAS 2002 Symposium, Vol. 1, pp 169-171 11

  7. Abstract • On-chip integration of isothermal PCR and electrophoresis • Loop-mediated isothermal amplification(LAMP) • Novel DNA amplification method developed by Eiken Chemical Co., Ltd. • By using 4 different primers specifically designed to recognize six distinct regions on the target gene • Performed at a constant temperature using a strand displacement reaction. • Advantages • High amplification efficiency( 109-1010 times in 15-60 minutes) • High specificity • The potential to replace PCR» simplicity, rapidity, specificity and cost-effectiveness • Avoid the thermal cycling • Analysis • by the integrated microchip electrophoresis • by the naked eyes by adding fluorescent dye like SYBR Green I 12

  8. Loop-mediated Isothermal Amplification (LAMP) 13

  9. RAM(Ramplification Amplification Method) 14

  10. Chip Device and Electropherogram • Integrated microchip for DNA amplification and CE • Reaction condition • RAMP; 5- 10 ul volume, 50-65°C for 10-30min • Micro-CE - Reproducibility(Fig. 4) - Detection time; 10min (Fig 5) - Visualization(Fig 6) Fig 1. Structure of a PMMA chip and device and electropherogram 15

  11. SYBR Green™ I • The dye (ex; EtBr) • Binds preferentially to ds DNA & quantitate the amount of double-stranded product • Stable to the extremes of temperature required for PCR reaction 6

  12. The Reproducibility and Detection 16

  13. Conclusions • Integrated microsystem • Useful for fast, sensitive, and simple detection of specific sequence of DNA • Application - O-157 E.coli, B. anthracis, Salmonella and other bacteria in the environment, food, animal, human blood, etc. 17

  14. Further Study • Development of DNA Computing Chip • DNA hybridization, ligation, PCR, CE • the uTAS 2003 Symposium • Application of DNA Computing • Diagnosis, Gene expression, Environmental Checker etc… • uTAS 2002 Paper • To access the latest papers published in LOC please see http://www.rsc.org/is/journals/current/loc/lcadvarts.htm • For Advance articles http://www.rsc.org/is/journals/current/loc/lc002003.htm • The general website is www.rsc.org/loc 18

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