We propose further applications of kinetic PCR-based methods for accurate and reproducible nucleic acid quantitation. We have shown that the kRT-PCR (kinetically-monitored reverse transcription-initiated PCR) assay accurately quantitates transcript levels across the entire physiologic range of abundance using total cellular RNA as template and transcript- specific primer pairs. Similarly, we have evidence that k-PCR (kinetically- monitored PCR) can be used to accurately score single nucleotide polymorphisms (SNPs) directly, eliminating post-PCR processing with gels, hybridization, or ELISA. KTC reactions are robotically assembled and utilize a thermostable DNA polymerase. computer-controlled digital camera monitors the cyclewise kinetics of product accumulation by fluorescence. Digital image analysis provides for full computer data handling. initial template concentrations are computed from the kinetics of PCR product accumulation. Previous results show that the kRT-PCR assay quantitates transcript level differences between two physiological or genetic states within a factor of 20%. Absolute mRNA levels are quantitated by kRT-PCR assay within a factor of two. Single nucleotide primer/template mismatches cause a 6 to 12 PCR cycle delay in the kinetics of PCR product accumulation. The magnitude of this latter primer/template discrimination readily supports accurate SNP allele frequencies determinations using DNA-templated kinetic PCR assays. Experiments are proposed to increase throughput by further improvements to automation of reaction assembly, assay quality assurance, primer design, and data analysis. Approaches are proposed to develop a larger capacity KTC instrument for applications that require performing very large numbers of assays. The Experimental Plan also includes application of the kinetic RT-PCR assays for genetic dissection of transcription paths and regulatory networks within the yeast transcription factors and identification of regulatory paths from the yeast transcriptional factors to target genes. Finally, experiments are proposed to develop and assess the capability of kinetic PCR assays for SNP-based linkage or linkage disequilibrium analyses of human DNAs. The accuracy, high sensitivity, wide detection range, and flexibility of kinetic PCR assays enable this technology to address a wide range of genomics-based analyses.
