Quadruplex-Based Technology for Nucleic Acid Amplification and Detection

Abstract

Polymerase chain reaction (PCR) has impacted nearly every field in molecular biology, genetics and forensic science. Numerous applications of PCR have been described for basic scientific purposes, as well as for diagnosis of hereditary and infectious diseases. This is particularly true for real-time PCR (RT-PCR), which detects and quantifies product molecules within PCR reaction vessels. However, PCR is still considered a daunting task due to many variables in the reaction, temperature cycling and complicated quantification methods. Current RT-PCR specific probes (Molecular Beacons, TaqMan, Scorpions) require costly synthesis and considerable effort to achieve optimal sensitivity. Typically, a fluorophore-quencher pair is attached to the ends of a probe, which doesn’t fluoresce when free in solution. Upon probe hybridization to an amplicon, the fluorophore is separated from the quencher and a signal is released.Recently we have discovered that the free energy of DNA quadruplexes can be used to drive unfavorable (endergonic) reactions of nucleic acids (e.g., isothermal PCR). The key point of quadruplex-driven reactions is that some G-rich sequences are capable of forming quadruplexes with significantly more favorable thermodynamics than the corresponding DNA duplexes. The energy of quadruplex formation can be used to drive PCR at constant temperature or DNA signal amplification. In addition, fluorescent nucleotide analogs incorporated and fully quenched within the primers regain maximum emission upon quadruplex formation, allowing very simple and accurate detection of product DNA. Thermodynamic and fluorescent bases of quadruplex priming amplification will be discussed.Supported by Bill & Melinda Gates Foundation.