PALM-SIZED REAL-TIME RT-PCR SYSTEM.ppt 1

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Published on April 7, 2009

Author: sweetsathy

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PALM-SIZED REAL-TIME RT-PCR SYSTEM : PALM-SIZED REAL-TIME RT-PCR SYSTEM SATHYA SADHASIVAM 27 MAR 2009 Pavel Neuzil1, Lukas Novak2, Juergen Pipper3, Shinhan Lee4, Lisa F. P. Ng5 and Chunyan Zhan 1Institute of Microelectronics, SINGAPORE 2Czech Technical University, CZECH REPUBLIC 3Institute of Bioengineering and Nanotechnology, SINGAPORE 4Nanyang Polytechnic, SINGAPORE 5Genome Institute of Singapore, SINGAPORE Slide 2: Introduction PCR is a powerful method Generates copies of DNA by temperature cycling in the presence of an enzyme Slide 3: PCR Slide 4: Photograph of a portable real-time RT- PCR system in an aluminum housing A. The PCR mixture, encapsulated in a droplet of oil is placed on top of a thin film resistor/temperature sensor for thermo cycling. B. The optical fluorescence system is located underneath the PCR chip. PALM-SIZED REAL-TIME RT-PCR SYSTEM : PALM-SIZED REAL-TIME RT-PCR SYSTEM System is compact Total weight 150 g, diameter 100 mm, and power consumption 3 W Volume of sample is Between 100 nL and 5 µL; routinely being 1 µL. PCR is conducted on a disposable microscope glass cover slip. capable of detecting RNA ,DNA , infectious agents like the HPAI (H5N1) virus in 20 min by the SYBRGreen I-based RT-PCR. Slide 6: A PCR chip is integrated with a miniaturized fluorescence system. Other PCBs contain analog circuits for the PCR thermal management, fluorescence data processing, microcontroller, and a power supply. The high-performance chip-based thermocycler is manufactured by silicon micromachining. Center of the chip is hollow, thus allowing optical access to the sample from below the chip, where the miniaturized fluorescence detection system is placed. Slide 7: EXPERIMENTAL Sample with 1 µL of RT-PCR mixture was transferred to a microscope cover slip on top of the PCR thermocycler and covered with 3 µL mineral oil to prevent evaporation. R. transcription was performed at 61 °C for 5 min followed by a ‘hot start’ at 95 °C for 20 s 4 s at 95 °C Denaturation. 20 s at 50 °C Annealing. Total time required for a single cycle was 34 s. transition time from one tem to another was only a few sec. Thermal profile is nearly rectangular, which allows further optimization of the thermal protocol without compromising the PCR efficiency. RESULTS AND DISCUSSION : RESULTS AND DISCUSSION Raw fluorescent signal from the optical unit was recorded simultaneously with the temperature total time around 35 min. Fluorescence profile of the RT-PCR (A), followed by a melting curve analysis. The temperature profile of a single PCR cycle is shown as an inset (A), demonstrating fast heating and cooling rates. Corrected average fluorescenceextracted in the last 2 s during the 72 °C segment (b) Slide 9: CONCLUSIONS The high-performance chip-based thermocycler is manufactured by silicon micromachining. A micro liter-sized PCR sample, covered by mineral oil, is placed on a disposable microscope cover slip on the top of the silicon chip. This configuration prevents sample to sample cross contamination. Designed truly portable real-time RT-PCR for the detection of RNA or DNA of infectious agents

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