CHEMILUMINISCENT DETECTION OF T CELL PCR PRODUCTS

THE POLYMERASE CHAIN REACTION (PCR) IS A POWERFUL TOOL USED IN VIRTUALLY ALL ASPECTS OF MOLECULAR BIOLOGY. THIS RESEARCH EFFORT WILL BE TO DETECT PCR PRODUCTS USING A PRIMER LABELED WITH A FLUOROPHORE THAT IS SUBSEQUENTLY ACTIVATED THROUGH A CHEMILUMINESCENT (CL) REACTION. THE CL REACTION WILL EMPLOY ENERGY TRANSFER, WHICH IS CAPABLE OF ACTIVATING SEVERAL FLUROPHORE LABELS WITH THE SAME REACTION. THIS HAS THE POTENTIAL FOR SIMULTANEOUSLY ACTIVATING AND DETECTING SEVERAL DIFFERENT COLORED PCR PRODUCTS IN A SINGLE STEP. THE ADDITIONAL ADVANTAGES OF NON-RADIOACTIVITY AND SPEED SEEN WITH OTHER CL SYSTEMS SHOULD BE POSSIBLE. IN THIS PROJECT,THE PRIMER IS LABELED WITH FLOURESCEIN. THE LABELED PRIMER IS USED TO PRODUCE THE CORRESPONDING LABELED DNA USING PCR. THE PRODUCTS ARE SEPARATED BY GEL ELECTROPHORESIS AND TRANSFERRED TO A MEMBRANE. THE LABELED PRODUCT IS THEN DETECTED THROUGH A CL REACTION INITIATED BY THE ADDITION OF PEROXYOXALATE AND HYDROGEN PEROXIDE. THE LIGHT PRODUCED GIVES A DIRECT MEASUREMENT OF THE AMOUNT OF LABELED DNA PRESENT. IF SUCCESSFUL, DIFFERENT COLORED PCR PRODUCTS COULD BE DETECTED SIMULTANEOUSLY IN THE SAME PREPARATION. COLORED CL TESTING COULD DETECT MULTIPLE DNA PROBES FOR RAPID SCREENING OR DIAGNOSTIC TESTING.