Real-Time Quadruplex Priming Amplification (QPA) for Nucleic Acid Amplification and Quantification
An enhanced nucleic acid amplification system that is more efficient and cost-effective than conventional polymerase chain reaction methods.
Identification of a particular nucleic acid is desirable in the use of diagnostic and forensic applications. However, often the target nucleic acid sequence may only be a small portion of the DNA or RNA in question so that it might be difficult to detect the presence of the target sequence using probes. Processes like Polymerase Chain Reaction (PCR) amplification are used to amplify the target sequence so that it is present in sufficient quantities.
Real-time polymerase chain reaction (RT-PCR) is a diagnostic and quantification tool used by researchers in molecular biology, genetics, and forensic science. Current RT-PCR techniques have several drawbacks, which limit their diagnostic capabilities. Thermal cyclers are needed to constantly initiate denaturation and annealing of sequenced DNA, which is costly and can increase mis-priming of products and influence an unstable environment for polymerases. The yield of specified products is also limited in quantitative RT-PCR diagnostics due to the competition between primer binding and self-annealing of product. In addition, RT-PCR mechanisms are overly complex especially in consideration of the separate binding sites utilized for primer and probe sequences.
Dr. Besik Kankia and colleagues at The Ohio State University have developed an amplification system to reduce or eliminate the shortcomings of PCR and clone generation in sequencing for nucleic acids. In addition, they have pioneered a new method of immobilizing nucleic acids for identification.
Quadruplex DNA Amplification
Quadruplex priming amplification (QPA) uses universal guanine primers to create quadruplex nucleic acid sequences. Once formed, the free energy of the DNA quadruplexes drives endergonic reactions of nucleic acids. QPA eliminates DNA self-annealing and proceeds under nearly isothermal conditions. Additionally, fluorescent nucleotide analogs can be incorporated into the primers, eliminating the need for attachment of probe molecules. This technology is a sensitive, specific, robust, and inexpensive method of amplifying DNA and provides significant advantages over the current gold standard.
Isothermic Quadruplex Priming Amplification
In order to enhance their next generation sequencing method, Dr. Kankia refined quadruplex priming amplification to make it a completely isothermal process, which returns a 1010-fold amplification of a DNA signal in less than 40 minutes. By moving from a linear amplification process to a loop process, not only is an isothermal process possible, but the method can be used in detection of RNA pathogens without reverse transcription. Furthermore, this new sequencing method removes competition from self-annealing, since the process keeps the target region and extended end bound. Finally, with no use for complementary primers, the problem of primer dimers is eliminated.