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：（根据调查人员的摘要进行了改编）调查人员提议进一步发展高分辨率荧光融化曲线分析用于核酸。熔融曲线分析最方便地执行 PCR扩增后立即。产品可以通过特征性的熔融曲线，甚至可以区分单个基础变化。 Lightcycler（TM）是申请人的热循环的商业平台和荧光监测。调查人员将改变照明器达到慢速（0.01度 /秒）和稳定（+/- 0.01度）温度通过修改硬件，温度传感和控制机制来过渡。这种逐渐过渡接近平衡条件，并允许高分辨率熔解曲线，类似于吸收性测量。通过缓慢熔化获得的分辨率增加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/等位基因熔化的多路复用程度温度取决于熔融曲线的分辨率和温度稳定性。不匹配位置和探针长度对单基础不匹配的影响将研究与完美匹配的探针的歧视，以评估这种突变扫描方法的潜力。最后，高分辨率长DNA的熔融曲线将使用双链DNA染料获得与吸收技术相比，SYBR绿色I和熔融域。拟议的商业应用程序：不可用