Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, 3400 N. Charles Street, Baltimore, MD 21218, USA.
Small. 2013 Apr 8;9(7):1096-105. doi: 10.1002/smll.201202242. Epub 2012 Dec 13.
Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and a tedious assay processes. In this report, an assay technology is proposed which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single-molecule coincidence detection, and the superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single-molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification.
基因点突变是遗传疾病的重要生物标志物。然而，现有的点突变检测方法存在灵敏度、特异性低和繁琐的检测过程等问题。本报告提出了一种将缺口连接酶链反应（Gap-LCR）的出色特异性、单分子符合检测的高灵敏度和量子点（QD）的优异光学性能相结合的检测技术，用于对基因组 DNA 中的点突变进行多重检测。Gap-LCR 产生突变特异性连接产物，然后通过 QD 捕获形成 DNA-QD 纳米复合物，通过多色荧光爆发符合分析进行单分子光谱（SMS）检测，实现无分离的多重突变检测。该检测方法能够以近 100%的特异性检测出皮摩尔级的 KRAS 密码子 12 突变变体。其高灵敏度允许直接在未经 PCR 预扩增的粗基因组 DNA 中检测 KRAS 突变。
