Pyrene-labeled base-discriminating fluorescent DNA probes for homogeneous SNP typing
Pyrene-labeled base-discriminating fluorescent DNA probes for homogeneous SNP typing
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DOI：
10.1021/ja039625y
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发表时间：
2004-04-21
影响因子：
15
通讯作者：
Saito, I
中科院分区：
文献类型：
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作者：
Okamoto, A;Kanatani, K;Saito, I
本文介绍了新的碱基识别荧光（BDF）核碱基的设计及其应用于单核苷酸多态性（SNP）分型。我们设计了新的BDF核苷，U-Py和C-Py，其中包含一个芘甲酰胺生色团连接的炔丙基接头。含有U-Py/A碱基对的双链体的荧光光谱显示在327 nm激发下在397 nm处的强发射。相反，含有U-Py/N碱基对（N = C、G或T）的双链体的荧光相当弱。含有匹配的U-Py/A碱基对的双链体的拟议结构表明，芘甲酰胺基团附近的高极性是负责强A-选择性荧光发射。此外，含有U-Py/A碱基对的双链体的荧光不被侧翼的C/G碱基对淬灭。荧光性质与以前的BDF核碱基完全不同，其中荧光可通过侧翼C/G碱基对淬灭。当与C-Py相对的碱基为G时，含有C衍生物C-Py的双链体选择性地发射荧光。互补碱基性质引起的荧光强度的剧烈变化对于SNP分型极其有用。含U-Py和C-Py的寡脱氧核苷酸作为有效的报告探针，用于含c-Ha-ras和BRCA 2 SNP位点的DNA样品的同质SNP分型。
This paper describes the design of novel base-discriminating fluorescent (BDF) nucleobases and their application to single nucleotide polymorphism (SNP) typing. We devised novel BDF nucleosides, U-Py and C-Py, which contain a pyrenecarboxamide chromophore connected by a propargyl linker. The fluorescence spectrum of the duplex containing a U-Py/A base pair showed a strong emission at 397 nm on 327 nm excitation. In contrast, the fluorescence of duplexes containing U-Py/N base pairs (N = C, G, or T) was considerably weaker. The proposed structure of the duplex containing a matched U-Py/A base pair suggests that the high polarity near the pyrenecarboxamide group is responsible for the strong A-selective fluorescence emission. Moreover, the fluorescence of the duplex containing a U-Py/A base pair was not quenched by a flanking C/G base pair. The fluorescence properties are quite different from previous BDF nucleobases, where fluorescence is quenchable by flanking C/G base pairs. The duplex containing the C derivative, C-Py, selectively emitted fluorescence when the base opposite C-Py was G. The drastic change of fluorescence intensity by the nature of the complementary base is extremely useful for SNP typing. U-Py- and C-Py-containing oligodeoxynucleotides acted as effective reporter probes for homogeneous SNP typing of DNA samples containing c-Ha-ras and BRCA2 SNP sites.