基于核酸短暂杂交的基因突变检测方法研究

Precise point mutation detection is of great importance and significance for the early diagnosis and screening of cancer, the mechanism of tumor formation and growth as well as the strategy of medicine. Hybridization based probes have been widely used for known mutation detection, however, they are not sensitive for single nucleotide mutation due to the barrier of hybridization thermodynamics. Nucleic acid transient binding (NATB) is the equilibrium of association and dissociation of nucleic acid under melting temperature which is sensitive to single nucleotide mismatch and is breaking the barrier of hybridization thermodynamics. The length of the duplex is 9-12 nt under room temperature. NATB holds a great potential for super-resolution imaging and biochemical analysis. This project aims to promote the selectivity to single nucleotide variants (SNVs) by NATB through single molecule fluorescence approach and kinetic coupled reaction approach, finally achieving accurate detection of gene mutation in blood sample of cancer patients.

精准的基因突变检测技术对于肿瘤早期诊断、发病机制研究以及用药策略有着重要的意义。杂交型核酸探针被广泛应用于已知突变位点的检测，但是由于杂交热力学的限制，对于单碱基突变不敏感。核酸短暂杂交是指双链核酸在其解链温度附近呈现杂交与解链的平衡，在室温条件下，一般杂交的双链长度为9-12个碱基。这种杂交模式突破核酸稳定杂交的热力学瓶颈，对单碱基变化非常敏感，在超分辨成像和生化分析领域有较好的应用前景。本课题拟利用核酸短暂杂交，通过单分子荧光和匹配动力学反应的手段，提高检测方法对单碱基变化的敏感度，进而实现对肿瘤患者血液样品中的基因突变的准确检测。

基因突变检测对于肿瘤早期筛查和病理研究具有重要的意义。基于杂交型探针的方法具有快速简便的特点，但由于选择性差，对于单碱基突变不敏感，在实际应用中常造成假阳性或者假阴性信号。本项目开发了基于核酸短暂杂交的方法，从原理上创新，解决了杂交型探针选择性差的问题，同时利用单分子荧光、磁性纳米粒子、选择性核酸酶等手段提高了检测的灵敏度，成功用于临床血液样品中循环肿瘤核酸中基因突变的检测（包括肺癌和结直肠癌相关的EGFR、KRAS、BRAF等基因突变），目前正在开发相关试剂盒，有望在医院检验科进行试用。在本项目的支持下，共发表高水平SCI论文14篇，申请专利5项，其中授权1项。