Fluorescent Determination of SNPs by C-Bulge binding ligand

对单个基因组中的单核苷酸多态性（SNP）进行分型对于诊断疾病风险、预测药物治疗效果以及优化个性化医疗是必要的。最近，我们报道了N，N' -双（3-氨丙基）-2，7-二氨基-1，8-萘啶（DANP）是双链DNA中单个胞嘧啶和胸腺嘧啶凸起的特异性稳定剂。在中性pH值下，DANP与胞嘧啶结合，伴随着1，8-萘啶中氮原子的质子化。与胞嘧啶结合的质子化DANP（DANPH^+）在430 nm处发射荧光，该荧光从其未结合状态向更长波长移动30 nm。在与胞嘧啶凸起结合后，吸收光谱也从360 nm移至400 nm。410 nm的辐射使我们能够选择性地激发结合在胞嘧啶隆突上的DANPH^+，在存在具有C-balge的游离靶DNA的情况下。本文介绍了一种新的荧光单核苷酸多态性分型方法，该方法基于C-凸起结合配体结合C-凸起形成DNA探针5 ' -d（ACATCCAAXCAACCAC）-3'和17-mer C-探针5' -（GTGGTTGYCTTGGATGT）-3'，其中X和Y为任意核苷酸碱基。当C-探针与靶DNA杂交时，胞嘧啶（下划线）产生直接邻近X-Y碱基对的单个胞嘧啶凸起。通过交替靶链中的X（A、T、C、G）和C-探针链中的Y（A、T、C、G和肌苷（I）），制备具有侧接20个不同匹配和错配碱基对的单个胞嘧啶凸起的双链体。

Typing of the single nucleotide polymorphisms (SNPs) in an individual genome against sets of predetermined SNPs will be necessary for diagnosing the disease risks, predicting effects on drug treatments, and optimizing the personalized medicine. Recently, we have reported N, N' -bis(3-aminopropyl)-2, 7-diamino-1, 8-naphthyridine (DANP) is a specific stabilizer of a single cytosine and thymine bulge in duplex DNA. DANP binds to the cytosine bulge accompanied by the protonation of a nitrogen atom in the 1, 8-naphthyridine at a neutral pH. The protonated DANP (DANPH^+) bound to the cytosine emitted fluorescence at 430 nm, which was shifted by 30 nm to a longer wavelength from its unbound state. The absorption spectrum was also shifted from 360 to 400 nm upon binding to the cytosine bulge. Irradiation at 410 nm enables us selectively to excite the DANPH^+ bound to the cytosine bulge in the presence of free target DNA having C-balge. Here, we describe a new method of fluorescent SNP typing based on the C-bulge binding ligand combined with a C-balge forming DNA probes.5' -d(ACATCCAAXCAACCAC)-3' and 17-mer C-probe 5' -(GTGGTTGYCTTGGATGT)-3', where X and Y were any nucleotide bases. When C-probe was hybridized to the target DNA, the cytosine (under lined) produced a single cytosine bulge directly neighboring to the X-Y base pair. By alternating X (A, T, C, G) in the target strand and Y (A, T, C, G, and inosine(I)) in the C-probe strand, duplexes with a single cytosine bulge flanking 20 different matched and mismatched base pairs were prepared.