title: Homogeneous label-free genotyping of single nucleotide polymorphism using ligation-mediated strand displacement amplification with DNAzyme-based chemiluminescence detection.
abstract: Genotyping of single nucleotide polymorphisms (SNPs) is a central challenge in disease diagnostics and personalized medicine. A novel label-free homogeneous SNP genotyping technique is developed on the basis of ligation-mediated strand displacement amplification (SDA) with DNAzyme-based chemiluminescence detection. Discrimination of single-base mismatches is first accomplished using DNA ligase to generate a ligation product between a discriminant probe and a common probe. The ligated product then initiates two consecutive SDA reactions to produce a great abundance of aptamer sequences against hemin, which can be probed by chemiluminscence detection. The developed strategy is demonstrated using a model SNP target of cytochrome P450 monooxygenase CYP2C19*2, a molecular marker for personalized medicines. The results reveal that the developed technique displays superb selectivity in discriminating single-base mismatches, very low detection limit as low as 0.1 fM, a wide dynamic range from 1 fM to 1 nM, and a high signal-to-background ratio of 150. Due to its label-free, homogeneous, and chemiluminescence-based detection format, this technique can be greatly robust, cost-efficient, readily automated, and scalable for parallel assays of hundreds of samples. The developed genotyping strategy might provide a robust, highly sensitive, and specific genotyping platform for genetic analysis and molecular diagnostics.
authors.forename: Hong-Qi
authors.initials: HQ
authors.lastname: Wang
authors.forename: Wei-Yu
authors.initials: WY
authors.lastname: Liu
authors.forename: Zhan
authors.initials: Z
authors.lastname: Wu
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Tang
authors.forename: Xiang-Min
authors.initials: XM
authors.lastname: Xu
authors.forename: Ru-Qin
authors.initials: RQ
authors.lastname: Yu
authors.forename: Jian-Hui
authors.initials: JH
authors.lastname: Jiang
mesh_terms: D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D021881:DNA, Catalytic / Q000378:metabolism*; D015894:Genome, Human / Q000235:genetics; D005838:Genotype; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
chemical_list: D021881:DNA, Catalytic; D015345:Oligonucleotide Probes; C400322:cytochrome P-450 CYP2C subfamily; D003577:Cytochrome P-450 Enzyme System