Real-time detection of nucleic acid interactions by total internal reflection fluorescence
Real-time detection of nucleic acid interactions by total internal reflection fluorescence
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DOI：
10.1021/ac0206519
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发表时间：
2003-05-15
影响因子：
7.4
通讯作者：
Klapproth, H
中科院分区：
文献类型：
--
作者：
Lehr, HP;Reimann, M;Klapproth, H
本文介绍了一种用于荧光标记DNA微阵列实时分析的光学读出系统的研制。该系统是针对基因组学，农业和生命科学的研究应用，其中最先进的读出系统的终点检测不能提供足够的信息杂交过程。可以观察到分子从流动池中的液相到换能器表面上的固定化寡核苷酸的杂交过程。荧光染料的激发由半导体激光器实现，荧光发射由冷却的CCD相机收集。可以从图像中提取定量数据用于微阵列的分析。对于信号传导，使用全内反射原理。通过多次内反射布置，传感器芯片适于标准显微镜载玻片格式，并且实现了传感器表面的有效区域的均匀渐逝照明。利用该读出系统进行了应用测试。在真实的时间中观察Cy 5标记的30-mer单链寡核苷酸与完全互补的固定化链的杂交。动力学分析证明与记录的数据。记录了与固定的野生型突变体15-和17-mer寡核苷酸杂交的来自血色病患者样品的140-mer PCR产物的解链曲线，并且可以检测到单点突变。
This paper describes the development of an optical readout system for the real-time analysis of fluorescent-labeled DNA microarrays is described. The system is targeted toward research applications in genomics, agriculture, and life sciences, where the end-point detection of state-of-the-art readout systems does not provide sufficient information on the hybridization process. The hybridization progress of molecules from the liquid phase in a flow cell to immobilized oligonucleotides on a transducer surface can be observed. The excitation of fluorochromes is realized by a semiconductor laser, and the fluorescence emission is collected by a cooled CCD camera. Quantitative data can be extracted from the images for analysis of the microarray. For the signal transduction, the principle of total internal reflection is used. With a multiple internal reflection arrangement, the sensor chip was adapted to the standard microscope slide format and a homogeneous evanescent illumination of the active area of the sensor surface was achieved. An application measurement was carried out with this readout system. The hybridization of Cy5-labeled 30-mer single-stranded oligonucleotides to fully complementary immobilized strands was observed in real time. A kinetic analysis was demonstrated with the recorded data. Melting curves of a 140-mer PCR product from a hemochromatosis patient sample hybridized to immobilized wildtype mutant 15- and 17-mer oligonucleotides were recorded and single-point mutations could be detected.