Split molecular beacon for specific identification of cancer-related single nucleotide polymorphism

In the human genome, single-nucleotide polymorphisms (SNPs) can often lead to the activation of oncogenes and have a close association with tumor development or progression. Specific identification of SNPs and quantitative analysis of oncogenes could provide insight into the basis for early diagnosis and risk assessment of malignancy, as well as personalized medicine. Herein, we proposed a new biosensing platform to screen SNPs through the ligation/polymerization-mediated signaling of a split molecular beacon (SMB) where target-recycling benefits can be achieved. The SMB consists of two fragments, one of which contains a fluorophore group and the other was modified with a quencher group. In the absence of target DNA, the two fragments hybridize with each other and the fluorescence is quenched. After exposure to a wild-type target, they can be opened and ligated together by the ligase using the target as the template. Moreover, the primer is able to bind to target hybridization-released binding sites and initiate strand-displacement amplification (SDA), generating an enhanced signal. By combining the specificity of nick ligation with the signal amplification of target recycling, the human K-ras oncogene as a model target can be detected down to 8 pM. Moreover, a wide dynamic range (more than 3 orders of magnitude) and excellent ability to discriminate single-base mutations are achieved. Due to the homogeneous reaction and simple experimental steps, the target assay is greatly cost-efficient and readily automated. The desirable features and advantages make the proposed genotyping strategy a promising platform for genetic analysis and molecular diagnostics.