title: A self-assembled deoxyribonucleic acid concatemer for sensitive detection of single nucleotide polymorphism.
abstract: Polymerase-free and label-free strategies for DNA detection have shown excellent sensitivity and specificity in various biological samples. Herein, we propose a method for single nucleotide polymorphism (SNP) detection by using self-assembled DNA concatemers. Capture probes, bound to magnetic beads, can joint mediator probes by T4 DNA ligase in the presence of target DNA that is complementary to the capture probe and mediator probe. The mediator probes trigger self-assembly of two auxiliary probes on magnetic beads to form DNA concatemers. Separated by a magnetic rack, the double-stranded concatemers on beads can recruit a great amount of SYBR Green I and eventually result in amplified fluorescent signals. In comparison with reported methods for SNP detection, the concatemer-based approach has significant advantages of low background, simplicity, and ultrasensitivity, making it as a convenient platform for clinical applications. As a proof of concept, BRAF(T1799A) oncogene mutation, a SNP involved in diverse human cancers, was used as a model target. The developed approach using a fluorescent intercalator can detect as low as 0.1 fM target BRAF(T1799A) DNA, which is better than those previously published methods for SNP detection. This method is robust and can be used directly to measure the BRAF(T1799A) DNA in complex human serum with excellent recovery (94-103%). It is expected that this assay principle can be directed toward other SNP genes by simply changing the mediator probe and auxiliary probes.
authors.affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wu
authors.forename: Junhua
authors.initials: J
authors.lastname: Chen
authors.forename: Zhiyuan
authors.initials: Z
authors.lastname: Fang
authors.forename: Chenchen
authors.initials: C
authors.lastname: Ge
authors.forename: Zhicheng
authors.initials: Z
authors.lastname: Xiang
authors.forename: Chuanyan
authors.initials: C
authors.lastname: Ouyang
authors.forename: Puchang
authors.initials: P
authors.lastname: Lie
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Xiao
authors.forename: Luxin
authors.initials: L
authors.lastname: Yu
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.forename: Lingwen
authors.initials: L
authors.lastname: Zeng
mesh_terms: D003116:Color; D003124:Colorimetry; D004247:DNA / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D008855:Microscopy, Electron, Scanning; D020641:Polymorphism, Single Nucleotide; D012015:Reference Standards
chemical_list: D004247:DNA
keywords: AP; CP; Deoxyribonucleic acid concatemers; MB; MP; PBS; Polymerase-free; SA; SNP; Single nucleotide polymorphism; T1; WT1; auxiliary probe; biotin-modified single-stranded DNA; bsDNA; capture probe; double-stranded DNA; dsDNA; magnetic bead; mediator probe; phosphate buffer saline; single nucleotide polymorphism; streptavidin; target DNA; wildtype DNA