Experimental investigation on working of PCR chip

Abstract

The possibility of performing fast and small-volume DNA amplification and analysis on a single chip has attracted great interest over the past few years. Polymerase Chain Reaction (PCR) is the most commonly used to amplify a specific region of a DNA strand. This amplification is brought about by thermal cycling which consists of a series of cycles of temperature changes, the highest and the most significant being the denaturation temperature of 95 degree Celsius. This Project looks into the setup of the thermal cycler, fabrication of PCR well layers by using polydimethylsiloxane (PDMS), the basic chip design with PDMS well layers using acrylic headspace and the making and advantages of reusable chips with rubber frames. Vacuum sample loading of the chip i.e. liquid sample being jetted into the headspace of the chip due to air pressure driven by the vacuum established in the headspace and wells was also looked into. Experiments were carried out both with the ink only, and also with ink, sealed with oil. The purpose of oil to prevent evaporation was also studied in detail. Investigative studies were carried out to check the effect of insufficient vacuuming, generation of foam, appropriate height of the headspace, filling uniformity and filling ratio, inefficient vacuum loading, air bubble generation and evaluation of liquid evaporation, particularly at denaturation temperature, among others. The main focus was however to conduct experiments to study the reasons and different scenarios of sample evaporation from a chip. The effect of using different types of oil and another means to prevent condensation of the sample liquid onto the top surface of the chip, i.e. using ITO glass heater was elaborately studied. The reason to conduct the above analysis was to study the ways and practices already investigated by several authors individually to prepare a successful chip. This project puts together all these best practices and comes out with a series of check list cum factors to prepare a working chip and present to the reader, means of incorrect as well as correct operation of successful chip making and working which is necessary for DNA analysis.