The Development of a Novel Capillary Loop Convective Polymerase Chain Reaction Container—Ultrasonic Machining Reagent Container

• Main Authors: Shih-Chun Kao, 高士峻
• Other Authors: Ping-Hei Chen
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/g55mz7

碩士 === 國立臺灣大學 === 機械工程學研究所 === 106 === Capillary Loop Convective Polymerase Chain Reaction (CLCPCR) is a new application of convective PCR. It can complete the temperature cycle of PCR with a single heater. By heating the bottom of the loop container, the fluid would form a Rayleigh–Bénard convection cell and generate a circulating flow continuously. The purpose of this thesis is developing a new manufacturing procedure for the reagent container and analyzing relevant factors to prove the possibility of the new container. A glass substrate is fabricated with the groove of loop-shaped with an ultrasonic machining approach. The fabricated glass substrate is bonded with a top glass cover with the silicone glue to form the test piece. Last, we remove toxic materials to DNA in the container by using the cleaner and the autoclave. To ensure the fluid field and the temperature distribution in the container suitable for PCR, we inject a solution of PSP into the container to make flow visualization and put thermocouples into the container to do the temperature measurement. In the condition of the heater temperature at 130 ℃ and the surrounding temperature at 25 ℃, the highest and the lowest temperature measured in the container are in the temperature range of PCR thermocycling. Meanwhile, we discuss the effect of the roughness to bubble formation during the heating process and check which material may affect DNA amplification by biocompatible tests. The experimental results show that the HBV template with initial concentration 107 copies/ ml can be amplified in the container made by ultrasonic machining at present. It costs 180 μL of reagent each time and takes 25 minutes to amplify DNA. Now the reagent container can be produced in specification and we have eliminated the doubt about reproducibility of the old containers.