Friedrich Achim, Hoheisel Jörg D, Marmé Nicole, Knemeyer Jens-Peter
Department of Functional Genome Analysis, German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany.
FEBS Lett. 2007 Apr 17;581(8):1644-8. doi: 10.1016/j.febslet.2007.03.031. Epub 2007 Mar 20.
This article presents a new, highly sensitive method for the identification of single nucleotide polymorphisms (SNPs) in homogeneous solutions using fluorescently labeled hairpin-structured oligonucleotides (smart probes) and fluorescence single-molecule spectroscopy. While the hairpin probe is closed, fluorescence intensity is quenched due to close contact between the chromophore and several guanosine residues. Upon hybridization to the respective target SNP sequence, contact is lost and the fluorescence intensity increases significantly. High specificity is achieved by blocking sequences containing mismatch with unlabeled oligonucleotides. Time-resolved single-molecule fluorescence spectroscopy enables the detection of individual smart probes passing a small detection volume. This method leads to a subnanomolar sensitivity for this single nucleotide specific DNA assay technique.
本文介绍了一种全新的、高灵敏度的方法，用于在均相溶液中使用荧光标记的发夹结构寡核苷酸（智能探针）和荧光单分子光谱法鉴定单核苷酸多态性（SNP）。当发夹探针关闭时，由于发色团与几个鸟苷残基紧密接触，荧光强度会淬灭。与各自的目标SNP序列杂交后，接触消失，荧光强度显著增加。通过用未标记的寡核苷酸封闭含有错配的序列来实现高特异性。时间分辨单分子荧光光谱法能够检测单个智能探针通过一个小检测体积。这种方法为这种单核苷酸特异性DNA检测技术带来了亚纳摩尔级的灵敏度。
