KTC: AN ACCURATE METHOD FOR TRANSCRIPTOME ANALYSIS

我们建议进一步应用基于动态pcr的方法进行准确和可重复的核酸定量。我们已经证明，使用细胞总RNA作为模板和转录特异性引物对，kRT-PCR（动态监测逆转录启动PCR）测定准确地定量了整个生理丰度范围内的转录水平。同样，我们有证据表明，k-PCR（动态监测PCR）可以直接准确地对单核苷酸多态性（snp）进行得分，从而消除了用凝胶、杂交或ELISA进行的PCR后处理。KTC反应是机器人组装和利用耐热DNA聚合酶。计算机控制的数码相机通过荧光监测产品积累的循环动力学。数字图像分析提供了全计算机数据处理。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测定在两种生理或遗传状态之间的转录物水平差异在20%以内。绝对mRNA水平是定量的kRT-PCR测定在一个因素的两个。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周期延迟。后一种引物/模板区分的幅度很容易支持使用dna模板化动力学PCR测定准确的SNP等位基因频率。实验建议通过进一步改进反应装配自动化，分析质量保证，引物设计和数据分析来提高通量。提出了开发更大容量KTC仪器的方法，用于需要进行大量分析的应用。实验计划还包括应用动态RT-PCR分析对酵母转录因子的转录途径和调控网络进行遗传解剖，并鉴定酵母转录因子到靶基因的调控途径。最后，提出了实验来开发和评估动态PCR分析的能力，用于基于snp的连锁或连锁不平衡分析人类dna。准确性，高灵敏度，宽检测范围，以及动态PCR分析的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.