Label-free hybridization detection of a single nucleotide mismatch by immobilization of molecular beacons on an agarose film

We developed a new technique to immobilize a set of molecular beacons on an agarose film-coated slide and found that it has the ability to identify a single nucleotide difference in label-free DNA targets. The annealing properties, specificity and hybridization dynamics of the present technique were compared with those of the conventional technique that directly immobilizes molecular beacons on a planar glass slide. It is demonstrated that the molecular beacon array on an agarose film has high quench efficiency, an excellent discrimination ratio for single nucleotide mismatches and a short detection time. We hypothesize that such a low fluorescence background and high specificity molecular beacon array will find practical applications in label-free, high-throughput mutation analysis and disease diagnosis.

本研究开发了一种将分子信标（molecular beacon）固定于琼脂糖覆膜玻片（agarose film-coated slide）的新型技术，并发现该技术可实现无标记DNA靶标（label-free DNA targets）中单核苷酸差异的识别。本研究将该技术的退火特性、特异性与杂交动力学，与直接将分子信标固定于平面玻片（planar glass slide）的传统技术进行了对比。实验结果表明，琼脂糖覆膜玻片上的分子信标阵列具备较高的淬灭效率（quench efficiency）、优异的单核苷酸错配（single nucleotide mismatches）区分率，且检测时长更短。本研究推测，这种低荧光背景、高特异性的分子信标阵列，有望在无标记高通量（high-throughput）突变分析（mutation analysis）与疾病诊断领域实现实际应用。