Quadruplex Priming Amplification (QPA) for Nucleic Acid Diagnostics

Abstract

Due to its incredible sensitivity, polymerase chain reaction (PCR) is a method of choice for nucleic acid diagnostics. However, PCR is a complex reaction due to (1) limited yield of product DNA (PCR plateau), (2) temperature cycling, and (3) complicated quantification methods. We have developed quadruplex priming amplification (QPA) to greatly simplify nucleic acid amplification and real-time quantification assays. The method relies on specifically designed guanine-rich primers, which after polymerase elongation are capable of spontaneous dissociation from target sites and forming DNA quadruplex. The quadruplex is characterized by significantly more favorable thermodynamics than the corresponding DNA duplexes. As a result, target sequences are accessible for the next round of priming, and DNA amplification proceeds under isothermal conditions with improved product yield. In addition, the quadruplex formation is accompanied by an increase in intrinsic fluorescence of the primers, allowing simple and accurate detection of product DNA. The chapter discusses the thermodynamic and optical principles of QPA and its application in nucleic acid diagnostics.