Department of Biomedical Sciences and Engineering, National Central University, Jhong-Li 320, Taiwan; Department of Chemical and Materials Engineering, National Central University, Jhong-Li 320, Taiwan.
Department of Chemical and Materials Engineering, National Central University, Jhong-Li 320, Taiwan.
Biosens Bioelectron. 2018 Jan 15;99:170-175. doi: 10.1016/j.bios.2017.07.052. Epub 2017 Jul 25.
An implementation of neutralized chimeric DNA oligomer as a probe for sensitive detection of single nucleotide polymorphisms (SNPs) on a surface plasmon resonance imaging sensor is investigated. The chimeric DNA oligomer was synthesized in a conventional DNA synthesizer, containing neutral nucleotides with a methylated phosphate group. The secondary structures and melting points of the chimeric DNA fragment and its complexes with perfect-matched and single-mismatched complementary DNA molecules were examined by using circular dichroism and UV-vis spectroscopy in comparison with the native probe DNA counterpart. The results indicate that the chimeric DNA complexes can form a B-form structure and exhibit high thermostability. Moreover, the hybridization and discrimination efficiency of the chimeric probe DNA for the SNP genotyping were verified by using the SPRi sensor under different experimental conditions. The data reveal the effects of the ionic strength and operation temperature on the selectivity of the chimeric probe DNA for the SNP detection. The hybridization condition with a low ionic strength and high temperature allows the chimeric probe DNA distinguishing perfect-matched and single-mismatched target DNA molecules to the best extent, likely due to the reduced electrostatic repulsive force and presence of the additional methyl group on the backbone. Consequently, the direct and label-free detection with the SPR technique and neutralized chimeric probe DNA can be realized for the SNP genotyping by optimizing the operation condition and sequence design.
一种中性嵌合 DNA 寡聚物探针在表面等离子体共振成像传感器上用于灵敏检测单核苷酸多态性 (SNP) 的方法得到了研究。该嵌合 DNA 寡聚物在常规 DNA 合成仪中合成，其中包含带有甲基磷酸基团的中性核苷酸。通过圆二色性和紫外-可见光谱法，与天然探针 DNA 相比，研究了嵌合 DNA 片段及其与完全匹配和单错配互补 DNA 分子复合物的二级结构和熔点。结果表明，嵌合 DNA 复合物可以形成 B 型结构，并表现出较高的热稳定性。此外，还通过不同实验条件下的 SPRi 传感器验证了嵌合探针 DNA 对 SNP 基因分型的杂交和区分效率。数据显示了离子强度和操作温度对嵌合探针 DNA 用于 SNP 检测的选择性的影响。在低盐度和高温的杂交条件下，嵌合探针 DNA 能够最大限度地区分完全匹配和单错配靶 DNA 分子，这可能是由于静电斥力的降低和主链上额外甲基的存在。因此，可以通过优化操作条件和序列设计，利用 SPR 技术和中性嵌合探针 DNA 实现 SNP 基因分型的直接、无标记检测。
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