Optimisation of real time PCR experiments for quantification of nucleic acids and genotyping using hydrolysis probes

A number of real time PCR approaches have been published in the literature. In this thesis, the suitability of different real time PCR approaches using hydrolysis probes have been evaluated regarding PCR performance, cost effectiveness as well as handling. The effect of double-quenched probes as well as the impact of the increase of relative Flap endonuclease amount in quantitative real time PCR has been examined. In terms of genotyping a TaqMan™ assay, considered to be the gold-standard in this application, has been tested and compared to phosphorothioate modified probes, allele specific primers, SNAKE primers, an allele specific probe and primer assays as well as an assay using minor groove binder probes. Promising observations have been made in the case of double-quenched probes, phosphorothioate modified probes, SNAKE primers as well as minor groove binder probes