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：（改编自研究者的摘要）研究者建议进一步发展高分辨率荧光熔解曲线分析对于核酸。最方便地进行熔解曲线分析 PCR 扩增后立即进行。产品可通过以下方式识别特征熔解曲线甚至可以区分单个碱基的变化。 LightCycler (TM) 是申请人的热循环商业平台和荧光监测。调查人员将把 LightCycler 改造为实现缓慢（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来实现转换。这种逐渐转变接近平衡条件并允许高分辨率熔解曲线，类似于通过吸光度获得的熔解曲线测量。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。探针/等位基因熔解的多重程度温度取决于熔解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美匹配的探针的区别，以评估该方法用于突变扫描的潜力。最后，高清使用双链 DNA 染料可获得长 DNA 的熔解曲线 SYBR Green I 和熔解域与吸收技术的比较。拟议的商业应用：不可用