Fluorescent Determination of SNPs by C-Bulge binding ligand

个体基因组中单核苷酸多态性（SNPs）的分型对诊断疾病风险、预测药物治疗效果和优化个性化医疗是必要的。最近，我们报道了N， N' -双（3-氨基丙基）- 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下，DANP与1,8 -萘啶中的一个氮原子质子化并与胞嘧啶凸起结合。与胞嘧啶结合的质子化DANP （DANPH^+）在430 nm处发出荧光，该荧光从其未结合状态向更长的波长移动了30 nm。与胞嘧啶突起结合后，吸收光谱也从360 nm转移到400 nm。在410 nm的辐照下，我们可以选择性地激发与胞嘧啶突起结合的DANPH^+。在这里，我们描述了一种基于c -凸起结合配体结合c -凸起形成DNA探针的荧光SNP分型新方法。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探针与目标DNA杂交时，胞嘧啶（下面划线）产生紧邻X-Y碱基对的单个胞嘧啶凸起。通过交替靶链上的X （A， T， C， G）和C-探针链上的Y (A， T， C， G，和肌苷(I))，制备了20个不同匹配和不匹配碱基对两侧具有单个胞嘧啶凸起的双链。

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.