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HIGH RESOLUTION FLUORESCENT MELTING CURVE ANALYSIS

高分辨率荧光熔解曲线分析

基本信息

批准号：
6014920
负责人：
PHILIP S BERNARD
金额：
$ 10万
依托单位：
BIOFIRE DEFENSE, LLC.
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-07-01 至 2001-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6014920
关键词：
biomedical equipment development fluorescence fluorescence resonance energy transfer nucleic acid denaturation nucleic acid hybridization nucleic acid probes polymerase chain reaction

项目摘要

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

描述：（改编自研究者摘要）研究者建议进一步开发高分辨率荧光熔解曲线分析 for nucleic核酸.熔解曲线分析是最方便的 PCR扩增后立即进行。产品可通过以下方式识别：特征解链曲线，甚至可以区分单个碱基变化。 LightCycler（TM）是申请人用于热循环的商业平台和荧光监测。研究人员将改变LightCycler，实现缓慢（0.01度/秒）和稳定（+/-0.01摄氏度）温度通过修改硬件、温度传感和控制机制实现过渡。这种逐渐过渡接近平衡条件，高分辨率熔解曲线，与吸光度法获得的曲线相似测量.通过缓慢熔化获得的增加的分辨率将是通过杂交探针和荧光共振能量转移证明在模型系统中。通过探针/等位基因解链的多重化程度温度取决于解链曲线分辨率和温度稳定性。错配位置和探针长度对单碱基错配的影响将研究与完全匹配探针的区分，以评估这种方法用于突变扫描的潜力。最后，高分辨率用双链DNA染料获得长DNA的解链曲线 SYBR绿色I和熔融域与吸光度技术的比较。拟议商业应用：不可用