Quadruplex Priming Amplification of DNA Coupled with Nicking Endonuclease Activity

Abstract

Identification, amplification and quantification of nucleic acids for detection of pathogenic organisms and genetic diseases is one of the most important field in biomedical research. Polymerase chain reaction (PCR) is commonly used for nucleic acid diagnostics. However, temperature cycling, limited yield of product DNA, the need for specialized instrumentation and expensive detection probes are not compatible with the goals of point-of-care diagnostics. To address these limitations we have developed quadruplex priming amplification (QPA), which relies on specifically designed guanine-rich primers. After polymerase elongation, the primers are capable of spontaneous dissociation from the target sites and formation of a DNA quadruplex. As a result, target sequences are accessible for the next round of priming and DNA amplification proceeds under isothermal conditions. QPA can proceed in either a linear or an exponential amplification mode. Experiments here demonstrate that linear QPA has a potential to be highly specific. We have combined linear QPA with nicking enzymes to reach the level of exponential signal amplification. In our assays we use specifically designed probes that are complementary to both pathogen and QPA template. When the probe is annealed to the pathogen DNA and the nicking site is folded, the enzyme nicks the pathogen strand and creates the 5’-overhang or primer/template complex, which is elongated by polymerase. At this point the nicking enzyme and polymerase initiate QPA template production, which later binds to QPA primers and produces signal. We will discuss thermodynamic and kinetic bases of these multi-step reaction, which can be conducted isothermally between 55 and 65 °C. Supported by Bill & Melinda Gates Foundation.