title: Single nucleotide polymorphism discrimination with and without an ethidium bromide intercalator.
abstract: Single nucleotide polymorphism (SNP) genotyping is an important aspect in understanding genetic variations. Here, we discriminate SNPs using toe-hold mediated displacement reactions. The biological target is an 80 nucleotide long double-stranded-DNA from the mtDNA HV1 region, associated with maternal ancestry. This target has been specially designed with a pendant toehold and a cationic fluorophore, ATTO 647N, as a reporter, produced in a polymerase chain reaction. Rates of reaction for the toehold-polymerase chain reaction products (TPPs) with their corresponding complementary displacing sequences, labelled with a Black Hole Quencher 1, followed the order TPP-Cytosine > TPP-Thymine > TPP-Adenine ≥ TPP-Guanine. Non-complementary rates were the slowest with mismatches involving cytosine. These reactions, operating in a static/or contact mode, gave averaged readouts between SNPs within 15 min (with 80-90% quenching), compared to 25-30 min in previous studies involving fluorescence resonance energy transfer. Addition of an intercalating agent, ethidium bromide, retarded the rate of reaction in which cytosine was involved, presumably through stabilization of the base pairing, which resulted in markedly improved discrimination of cytosine containing SNPs.
authors.affiliation: Flinders Centre for Nanoscale Science and Technology, Flinders University, Sturt Road, Bedford Park, Adelaide, South Australia 5042, Australia.
authors.forename: Renzo A
authors.initials: RA
authors.lastname: Fenati
authors.affiliation: Flinders Centre for Nanoscale Science and Technology, Flinders University, Sturt Road, Bedford Park, Adelaide, South Australia 5042, Australia.
authors.forename: Ashley R
authors.initials: AR
authors.lastname: Connolly
authors.affiliation: Flinders Centre for Nanoscale Science and Technology, Flinders University, Sturt Road, Bedford Park, Adelaide, South Australia 5042, Australia; Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, VIC 3010, Australia. Electronic address: amanda.ellis@flinders.edu.au.
authors.forename: Amanda V
authors.initials: AV
authors.lastname: Ellis
mesh_terms: D004252:DNA Mutational Analysis; D004272:DNA, Mitochondrial / Q000032:analysis*; D004996:Ethidium / Q000737:chemistry*; D006801:Humans; D007364:Intercalating Agents / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide
chemical_list: D004272:DNA, Mitochondrial; D007364:Intercalating Agents; D004996:Ethidium
keywords: Fluorescence quenching; Genotyping; Kinetics; Nucleotide polymorphism; Strand displacement