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Quadruplex priming amplification coupled with EXPAR for point-of-care diagnostics

Research Scholar

Tamar Partskhaladze, Chemistry (Georgia)
Shota Gogichaishvili, Co-Researcher
Besik Kankia, Faculty Mentor

Biography

Tamar Partskhaladze graduated from Tbilisi State University, Georgia in 2004. She then continued work in the Laboratory of Biophysics as an engineer while completing her PhD thesis, entitled “pH-influence on DNA structural lability and its possible role in DNA-protein recognition.” During this period, she participated in various international conferences in countries such as Spain, Italy and Sweden. She defended her PhD thesis at the Faculty of Exact and Natural Sciences in Georgia in 2010, then joined Faculty of Medicine at Tbilisi State University as a lecturer on medical physics and biophysics.

What is the issue or problem addressed in your research?

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), which allows amplification of DNA from specimens containing minute quantities of a nucleic acid target, 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 goals of point-of-care (POC) diagnostics.

What methodology did you use in your research?

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 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 by fluorescence measurements. QPA can proceed in either a linear or an exponential amplification mode. Experiments here demonstrate that linear QPA is simpler and has a potential to be highly specific. Therefore, in the present work we combined linear QPA with EXPAR to reach level of exponential signal amplification. EXPAR relies on a combination of polymerase elongation and single strand nicking, and rapidly amplifies short DNA segments of interest.

What are the purpose/rationale and implications of your research?

Most nucleic-acid amplification reactions exhibit non-specific amplification in the absence of the targeted sequence, which limits possibility of application in molecular diagnostics. In our investigation different experimental conditions (solvent, temperature, template oligonucleotide concentrations) were examine to achieve maximal time difference between positive, specific amplification and negative, non-specific amplification reactions.