Quadruplex Priming Amplification at a Range of Human Body Temperature

Abstract

Quadruplex priming amplification (QPA) is a very simple amplification assay in which the isothermal amplification is performed using an only DNA polymerase, and detection is conducted by the intrinsic fluorescence of the primers. QPA employs specific G-rich sequences as primers that, upon polymerase elongation at specific temperatures, spontaneously dissociate from the primer binding sites (PBS) and fold into a monomolecular quadruplex. Fluorescent nucleotide analogs, such as 3-methyl isoxanthopterin (3MI), when incorporated into these primers, emit light upon a quadruplex formation and permit simple, specific, and sensitive quantification without the attachment of probe molecules. Previously has developed QPA assays with truncated targets and potassium cations that demonstrate an optimal amplification around 55°C. Here, we designed QPA assays with truncated target and led cations at a range of human body temperature. Lead cations reveal the most rapid amplification than potassium and strontium cations. QPA can be applied as a method for the implementation of simple and cheap diagnostics (point of care (POC)), as well as development at a range of 35-45°C temperature, which will make this method more convenient for using it in molecular diagnostics.