A dynamic sandwich assay on magnetic beads for selective detection of single-nucleotide mutations at room temperature.
Single-nucleotide mutation (SNM) has proven to be associated with a variety of human diseases. Development of reliable methods for the detection of SNM is crucial for molecular diagnosis and personalized medicine. The sandwich assays are widely used tools for detecting nucleic acid biomarkers due to their low cost and rapid signaling. However, the poor hybridization specificity of signal probe at room temperature hampers the discrimination of mutant and wild type. Here, we demonstrate a dynamic sandwich assay on magnetic beads for SNM detection based on the transient binding between signal probe and target. By taking the advantage of mismatch sensitive thermodynamics of transient DNA binding, the dynamic sandwich assay exhibits high discrimination factor for mutant with a broad range of salt concentration at room temperature. The beads used in this assay serve as a tool for separation, and might be helpful to enhance SNM selectivity. Flexible design of signal probe and facile magnetic separation allow multiple-mode downstream analysis including colorimetric detection and isothermal amplification. With this method, BRAF mutations in the genomic DNA extracted from cancer cell lines were tested, allowing sensitive detection of SNM at very low abundances (0.1-0.5% mutant/wild type).
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.
Junxiu
J
Wang
Department of Nephrology, Shenzhen Affiliated Hospital, Guangzhou University of Chinese Medicine, Shenzhen, Guangdong 518033, China.
Guoliang
G
Xiong
Clinical Laboratory, China-Japan Friendship Hospital, Beijing 100029, China.
Liang
L
Ma
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.
Shihui
S
Wang
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.
Xu
X
Zhou
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.
Lei
L
Wang
College of Chemistry, Nankai University, Tianjin 300071, China.
Lehui
L
Xiao
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China. Electronic address: xinsu@mail.buct.edu.cn.
Xin
X
Su
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China. Electronic address: yucy@mail.buct.edu.cn.
Changyuan
C
Yu
D015374:Biosensing Techniques / Q000379:methods*; D003124:Colorimetry / Q000379:methods*; D005838:Genotype; D006801:Humans; D060526:Magnetic Fields; D009154:Mutation / Q000941:ethics* / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry / Q000235:genetics*; D013696:Temperature
C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
Cancer biomarker detection; Magnetic beads; SNM; Transient DNA binding