Synthesis of Thermostable DNA Polymerase/Fluorescent Protein Chimeras for PCR Study

Abstract

The Polymerase Chain Reaction (PCR) is an invaluable technique in modern biochemistry that uses DNA polymerase enzymes from extremophile microbe species together with thermocycling to amplify a specific sequence of DNA. Another revolution in biochemistry is the use of fluorescent proteins which are reporter molecules of varying hues and intensities. This research combines PCR enzymes and fluorescent proteins. We have found a few fluorescent proteins that exhibit thermostability, refolding properties, or both. We plan to combine PCR polymerase enzymes with a fluorescent protein resistant to high temperature inactivation, specifically tdTomato or ZsGreen. A successful chimera can be used to elucidate the reaction taking place as well as a possible optimization of the processes involved. Four polymerase/fluorescent protein combinations were created with one chimera showing fluorescent activity.