Department of Chemical and Materials Engineering, National Central University, Jhong-Li, 32001, Taiwan.
Department of Bioinformatics and Medical Engineering, Asia University, Taichung, 41354, Taiwan.
Sci Rep. 2019 Jul 30;9(1):11056. doi: 10.1038/s41598-019-47522-9.
Silicon nanowire (SiNW) field-effect transistors (FETs) is a powerful tool in genetic molecule analysis because of their high sensitivity, short detection time, and label-free detection. In nucleic acid detection, GC-rich nucleic acid sequences form self- and cross-dimers and stem-loop structures, which can easily obtain data containing signals from nonspecific DNA binding. The features of GC-rich nucleic acid sequences cause inaccuracies in nucleic acid detection and hinder the development of precision medicine. To improve the inaccurate detection results, we used phosphate-methylated (neutral) nucleotides to synthesize the neutralized chimeric DNA oligomer probe. The probe fragment originated from a primer for the detection of hepatitis C virus (HCV) genotype 3b, and single-mismatched and perfect-matched targets were designed for single nucleotide polymorphisms (SNP) detection on the SiNW FET device. Experimental results revealed that the HCV-3b chimeric neutralized DNA (nDNA) probe exhibited better performance for SNP discrimination in 10 mM bis-tris propane buffer at 25 °C than a regular DNA probe. The SNP discrimination of the nDNA probe could be further improved at 40 °C on the FET device. Consequently, the neutralized chimeric DNA probe could successfully distinguish SNP in the detection of GC-rich target sequences under optimal operating conditions on the SiNW FET device.
硅纳米线 (SiNW) 场效应晶体管 (FET) 因其高灵敏度、短检测时间和无标记检测而成为基因分子分析的有力工具。在核酸检测中，富含 GC 的核酸序列形成自和交叉二聚体以及茎环结构，这很容易从非特异性 DNA 结合中获得包含信号的数据。富含 GC 的核酸序列的特征导致核酸检测不准确，阻碍了精准医学的发展。为了提高不准确的检测结果，我们使用磷酸甲基化（中性）核苷酸来合成中性嵌合 DNA 寡核苷酸探针。探针片段来源于丙型肝炎病毒 (HCV) 基因型 3b 检测的引物，并且针对 SiNW FET 器件上的单核苷酸多态性 (SNP) 检测设计了单错配和完全匹配的靶标。实验结果表明，在 25°C 的 10 mM 双三丙撑基丙烷缓冲液中，HCV-3b 嵌合中性 DNA (nDNA) 探针在 SNP 区分方面的性能优于常规 DNA 探针。在 FET 器件上，nDNA 探针的 SNP 区分可以在 40°C 进一步提高。因此，中性嵌合 DNA 探针可以在 SiNW FET 器件上的最佳工作条件下成功区分富含 GC 的目标序列检测中的 SNP。
