title: Single-Molecule Counting of Point Mutations by Transient DNA Binding.
abstract: High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.
authors.affiliation: College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Su
authors.affiliation: College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.
authors.forename: Lidan
authors.initials: L
authors.lastname: Li
authors.affiliation: Institute of Quality Standard and Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Wang
authors.affiliation: College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.
authors.forename: Dandan
authors.initials: D
authors.lastname: Hao
authors.affiliation: College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.
authors.forename: Changyuan
authors.initials: C
authors.lastname: Yu
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D015342:DNA Probes / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D009693:Nucleic Acid Hybridization; D009711:Nucleotides / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism
chemical_list: D015342:DNA Probes; D004277:DNA, Single-Stranded; C117307:KRAS protein, human; D009711:Nucleotides; D016283:Proto-Oncogene Proteins p21(ras)