The success and sensitivity of DNA detection is limited in important clinical, forensic, and environmental applications due to the presence of complex biological samples, such as blood, which reduce amplification efficiency. Different pre-PCR treatments that are being used to overcome this inhibitory effect in crude specimens significantly increase the duration and cost of the tests. We propose to develop and functionally characterize Taq polymerase mutants resistant to the blood inhibitory effect, which would allow a faster and lower-cost PCR analysis of crude samples. We also plan to obtain mutants of Taq with rapid DNA elongation, which will reduce the time of the amplification cycle, and we will attempt to add this feature to the blood-resistant phenotype. The screening for the above mutants will be performed by directed evolution of mutagenized Taq enzyme, using a high throughput version of this strategy, through which thousands of mutant clones are simultaneously amplified in individual micro-compartments of an emulsion PCR mixture. This proposal was motivated by our initial data showing that the two desired features of Taq, high resistance to blood inhibition and faster DNA elongation, are indeed obtainable by mutagenesis. Finally, in a potential phase two of the project, we will try to combine these two novel qualities of Taq with the cold-sensitive mutants of the enzyme designed for hot-start PCR which have already been developed and marketed by our company.
