title: Isothermal quadruplex priming amplification for DNA-based diagnostics.
abstract: We previously developed a method, known as quadruplex priming amplification (QPA), which permits isothermal amplification of DNA. The assay is based on a DNA quadruplex formed by the GGGTGGGTGGGTGGG (G3T) sequence. G3T has three unique properties that are fundamental for QPA; (i) G3T forms a quadruplex with significantly more favorable thermodynamics than the corresponding DNA duplexes; (ii) removal of guanines at the 3'-end inhibits quadruplex formation; and (iii) incorporated fluorescent nucleotides, such as 2-aminopurine (2AP) or 6-methylisoxanthopterin (6MI), which are quenched by neighboring nucleotides, regain maximum emission upon quadruplex formation. New model studies carried out here with primers missing one, two and three guanines reveal that the driving force for QPA comes from the difference in thermal stability between the primer/template and the product complexes. Primers missing one and two guanines are able to self-dissociate from the template upon elongation, whereas QPA is not observed when the primer lacks three 3'-nucleotides. QPA reaches its maximum rate at temperatures slightly higher than the T(m) of the primer/template complex and is more efficient in the presence of only dGTP. QPA-based assays also revealed that Taq is able to incorporate thymidines opposite template 2AP, while no significant incorporation was observed opposite template 6MI.
authors.affiliation: Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Taylor
authors.forename: Anupama
authors.initials: A
authors.lastname: Joseph
authors.forename: Robert
authors.initials: R
authors.lastname: Okyere
authors.forename: Shota
authors.initials: S
authors.lastname: Gogichaishvili
authors.forename: Karin
authors.initials: K
authors.lastname: Musier-Forsyth
authors.forename: Besik
authors.initials: B
authors.lastname: Kankia
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry; D054856:G-Quadruplexes; D006147:Guanine / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013698:Templates, Genetic; D013816:Thermodynamics; D014976:Xanthopterin / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: C483770:6-methylisoxanthopterin; D017931:DNA Primers; D005456:Fluorescent Dyes; D015075:2-Aminopurine; D006147:Guanine; D004247:DNA; D014976:Xanthopterin