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Isothermal amplification and quantification of DNA using quadruplex primers with intrinsic fluorescence

Research Scholar

Shota Gogichaishvili, Chemistry and Biochemistry (Georgia)
Adam Taylor, Co-Reseacher
Anupama Joseph, Co-Researcher
Robert Okyere, Co-Researcher
Besik Kankia, Faculty Mentor

Biography

Shota Gogichaishvili graduated from Tbilisi State University in Georgia in 2004. He then worked in the Department of Biological System Physics at the Andronikashvili Institute of Physics as a science researcher while simultaneously working on his PhD thesis, “Supercooled water in the roots of annual plants.” He defended this thesis at the Faculty of Exact and Natural Sciences, Tbilisi State University in 2010.

What is the issue or problem addressed in your research?

Polymerase chain reaction (PCR) is a technique prevalent in research that is used to amplify small quantities of DNA. Current techniques require thermal cycling, which is both time consuming and expensive, and reach a plateau at low nanomolar concentrations of the product, due to competition between primer binding and self-annealing.

What methodology did you use in your research?

Quadruplex priming amplification (QPA) is a new technique that greatly simplifies conventional PCR by eliminating these problems. Special primers are designed such that, after transcription of two guanine molecules, the primer spontaneously folds into a quadruplex, exposing the primer binding site for another primer. Studies using primers labeled with the fluorescent nucleotide analogs 2-aminopurine (2Ap) and 6-methyl isoxanthopterin (6Mi) were performed in order to determine the optimal conditions for this reaction, including temperature, concentration, and composition of dNTP mixture, by measuring the rate of change in fluorescence over time. This quadruplex-based technique was then used for further studies of the mismatch incorporation abilities of the Taq and Bst polymerases. An assay was designed in which a change in fluorescence would only be observed if the quadruplex was allowed to fold by incorporation of a mismatch. Results from these assays indicate that Taq and Bst are severely limited in their abilities to incorporate mismatches.

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

Taken together, these results show the various ways in which quadruplex-based technologies could improve DNA detection and amplification.