Probes for detection of specific DNA sequences at the single-molecule level
Probes for detection of specific DNA sequences at the single-molecule level
复制标题
DOI：
10.1021/ac000024o
复制
发表时间：
2000-08-15
影响因子：
7.4
通讯作者：
Sauer, M
中科院分区：
文献类型：
--
作者：
Knemeyer, JP;Marmé, N;Sauer, M
已经开发了一种方法，用于在均相测定中使用标记的寡核苷酸分子结合单分子光子爆发计数和鉴定来高灵敏度检测特定DNA序列。荧光标记的寡核苷酸被称为智能探针，因为它们通过荧光强度的强烈增加来报告互补靶序列的存在。智能探针由连接在发夹寡核苷酸末端的荧光染料组成。所提出的技术利用了所使用的恶嗪染料JA 242被互补鸟苷残基有效猝灭的事实。在与靶DNA特异性杂交后，智能探针经历迫使荧光染料和鸟苷残基分开的构象变化，从而在整体测量中将荧光强度增加约6倍。为了增加检测灵敏度低于纳摩尔范围，共聚焦荧光显微镜用于观察在存在和不存在靶DNA的情况下，当它们通过聚焦激光束时来自各个智能探针的荧光爆发。智能探针由在635 nm处以64 MHz的重复率发射的脉冲二极管激光器激发。每个荧光猝发由三个独立参数识别：（a）猝发大小，（B）猝发持续时间，和（c）荧光寿命。通过使用这种多参数分析，实现了智能探针和杂交探针-靶双链体之间更高的区分准确度。所提出的多参数检测技术允许在均相测定中鉴定皮摩尔靶DNA浓度，即，检测过量200倍标记探针分子中的特定DNA序列。
A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide, The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 mn with a repetition rate of 64 MHz, Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe-target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules.