KTC: AN ACCURATE METHOD FOR TRANSCRIPTOME ANALYSIS

我们提出了进一步的应用动力学PCR为基础的方法，准确和可重复的核酸定量。我们已经证明，kRT-PCR（动力学监测的逆转录启动的PCR）测定法使用总细胞RNA作为模板和转录本特异性引物对准确定量整个生理丰度范围内的转录本水平。类似地，我们有证据表明，k-PCR（动力学监测PCR）可用于直接准确地对单核苷酸多态性（SNP）进行评分，消除了凝胶、杂交或ELISA的PCR后处理。KTC反应是机器人组装的，并利用热稳定的DNA聚合酶。 计算机控制的数字照相机通过荧光监测产物累积的循环动力学。数字图像分析提供完整的计算机数据处理。从PCR产物积累的动力学计算初始模板浓度。先前的结果表明，kRT-PCR测定定量两种生理或遗传状态之间的转录水平差异在20%的因子内。通过kRT-PCR测定在2倍内定量绝对mRNA水平。单核苷酸引物/模板错配导致PCR产物积累动力学中的6至12个PCR循环延迟。后一种引物/模板区分的幅度容易支持使用DNA模板化的动力学PCR测定的准确SNP等位基因频率测定。实验提出了通过进一步改进反应组装、测定质量保证、引物设计和数据分析的自动化来增加通量。提出了开发更大容量的KTC仪器的方法，用于需要执行非常大量的测定的应用。该实验计划还包括应用动态RT-PCR分析对酵母转录因子内的转录路径和调控网络进行遗传解剖，并鉴定从酵母转录因子到靶基因的调控路径。最后，实验提出了开发和评估的能力，基于SNP的连锁或连锁不平衡分析的人类DNA的动力学PCR检测。动态PCR检测的准确性、高灵敏度、宽检测范围和灵活性使该技术能够解决广泛的基于基因组学的分析。

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.