HIGH RESOLUTION FLUORESCENT MELTING CURVE ANALYSIS

描述：(改编自调查人员摘要)调查人员 建议进一步开展高分辨率荧光熔融曲线分析 对于核酸来说。最方便的是进行熔化曲线分析 扩增后即刻进行聚合酶链式反应。产品可以通过以下方式标识 即使是单碱基变化也能区分的特征熔融轮廓。 LightCycler(TM)是申请人用于热循环的商业平台 和荧光监测。调查人员将把LightCycler改装成 达到缓慢(0.01度/秒)和稳定(+/-0.01摄氏度)的温度 通过修改硬件、温度传感和控制机制实现过渡。 这种渐进的转变接近于平衡条件，并允许 高分辨率熔化曲线，类似于吸光度获得的曲线 测量。通过慢速熔化获得的更高分辨率将是 以杂交探针和荧光共振能量转移为例 在模型系统中。探针/等位基因融合的多重化程度 温度取决于熔化曲线的分辨率和温度稳定性。 不匹配位置和探针长度对单碱基不匹配的影响 将研究从完全匹配的探测器中进行区分，以评估 这种方法在突变扫描方面的潜力。最后，高分辨率 用双链DNA染料可以得到长DNA的熔融曲线 SYBR Green I和熔融结构域与吸光技术的比较。 建议的商业应用：不可用

DESCRIPTION: (Adapted from the investigator's abstract) The investigators propose to further develop high-resolution fluorescent melting curve analysis for nucleic acids. Melting curve analysis is most conveniently performed immediately after amplification by PCR. Products can be identified by characteristic melting profiles that can distinguish even single base changes. The LightCycler (TM) is the applicants' commercial platform for thermal cycling and fluorescent monitoring. The investigators will alter the LightCycler to achieve slow (0.01degrees /sec) and steady (+/- 0.01degrees C) temperature transitions by modifying hardware, temperature sensing, and control mechanisms. Such gradual transitions approach equilibrium conditions and allow high-resolution melting curves, similar to those obtained by absorbency measurements. The increased resolution obtained with slow melting will be demonstrated by hybridization probes and fluorescence resonance energy transfer in model systems. The degree of multiplexing by probe/allele melting temperatures depends on the melting curve resolution and temperature stability. The effect of mismatch position and probe length on single base mismatch discrimination from a perfectly matched probe will be studied to assess the potential of this method for mutation scanning. Finally, high-resolution melting curves of long DNA will be obtained with the double-stranded DNA dye SYBR Green I and the melting domains compared to absorbency techniques. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE