The growing markets for analytical techniques in areas such as pathogen detection, clinical analysis, forensic investigation, environmental analysis and food analysis require the development of devices with simultaneous high performance, speed, simplicity and low cost. Analysis of deoxyribonucleic acid (DNA) has been enhanced by use of the polymerase chain reaction (PCR) technique, which is now a widely used tool for in vitro amplification of nucleic acids. In this work, a miniaturised PCR system comprising a microfluidic PCR chip, novel heating method and fluorescence detection unit was developed. PCR chip with reactants were shunted along three temperature zones in a fine polycarbonate chip. The polycarbonate PCR chip was fabricated using milling and thermal fusion binding for sealing of the cover. Thermal-cycling within the microfluidic chip was achieved by programmable shunting of the chip between three double side temperature zones with different temperatures to accomplish the denaturation, annealing and elongation steps necessary for PCR amplification. This thermal-cycling model potentially improves PCR efficacy because it increases the ramping rates for heating and cooling the PCR mixture. The detection unit comprises a photo-detector and Light Emitting Diode (LED) as the source of excitation. The detection limit of the system was determined on the PCR chip using Fluorescein isothiocyanate (FITC) as a fluorophore dye. The detection limit achieved was 7.8 pg ml-1 or (19.7 pmol) of FITC. The chromosomal DNA used in this work was extracted from non-pathogenic K-12 subtype of Escherichia coli (E. coli). The investigations showed that the system was capable of performing PCR amplification with different annealing temperature ranging from 54 to 68 °C, targeting three different sizes of PCR products of 250, 552 and 1500 bp. The prototype thermal-cycler and PCR chip were used successfully to amplify the three sizes and the results were compared with same fragments amplified on a conventional PCR .thermal-cycler machine. The method used for comparison was gel electrophoresis. In addition, a fluorescence detection system was employed for detecting of PCR products using SYBR Green I fluorescent dye. The whole system allows for developments of low cost, easy to use and portable instruments.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:606218 |
| Date | January 2013 |
| Creators | Salman, Abbas Ali Abulwohab |
| Contributors | Carney, Helen |
| Publisher | Teesside University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10149/316214 |
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