Exponential Quadruplex Priming Amplification for DNA-based Isothermal Diagnostics Tamar Partskhaladze, 1 Adam Taylor, 1 Levan Lomidze, 1,2 David Gvarjaladze, 1,2 Besik Kankia 1,2 1 Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210 2 Institute of Biophysics, Ilia State University, Tbilisi 0162, Republic of Georgia Received 4 May 2014; revised 27 July 2014; accepted 14 August 2014 Published online 1 October 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22557 ABSTRACT: Polymerase chain reaction (PCR) is a method of choice for molecular diagnostics. However, PCR relies on ther- mal cycling, which is not compatible with the goals of point-of-care diagnostics. A simple strategy to turn PCR into an isothermal method would be to use specific pri- mers, which upon polymerase elongation can self- dissociate from the primer-binding sites. We recently demonstrated that a monomolecular DNA quadruplex, GGGTGGGTGGGTGGG, meets these requirements, which led to the development of the linear versions of quadruplex priming amplification (QPA). Here we dem- onstrate exponential version of isothermal QPA, which allows an unprecedented 10 10 -fold amplification of DNA signal in less than 40 min. VC 2014 Wiley Periodicals, Inc. Biopolymers 103: 88–95, 2015. Keywords: DNA quadruplexes; QPA; isothermal amplification This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the pre- print version. You can request a copy of any preprints from the past two calendar years by emailing the Biopolymers editorial office at biopolymers@wiley.com. INTRODUCTION P olymerase chain reaction (PCR), a method of choice for molecular diagnostics, relies on thermal cycling, consisting of repeated temperature changes for DNA unfolding, primer annealing and enzymatic replica- tion of the DNA template. 1,2 Temperature cycling, which requires specialized instrumentation and considerable power supply, is not compatible with the goals of point-of- care (POC) diagnostics. To avoid thermal cyclers, several iso- thermal amplification assays have been developed (as a review see Ref. [2). However, all of these methods employ complicated amplification mechanisms using extra protein and DNA components, which affect cost, sensitivity and/or specificity of the amplification. Currently, three main techniques, specific to the amplified product, are used to monitor real-time PCR: Molecular Bea- cons, 3,4 TaqMan 5 and Scorpion probes. 6 All specific probe molecules have similar disadvantages which include: (i) the requirement of two external and costly tags (fluorophore and quencher); (ii) the contribution of two separate functions in the probes: recognition and reporting (fluorescence), which makes the assay very expensive when several targets are tested; (iii) the use of separate binding sites for primer and probe sequences, which introduces another component (probe oligo- nucleotide) to an already complex reaction and adds additional design limitations due to the need to avoid interactions between the probe and primers; and (iv) the use of a bimolecu- lar probe-primer system, which makes the reaction entropically unfavorable, slowing down hybridization and complicating product detection at exponential growth. Reporting is much faster and more efficient with a monomolecular probe-primer system as proposed earlier. 6 Additional Supporting Information may be found in the online version of this article. Correspondence to: Besik Kankia; e-mail: kankia.1@osu.edu Contract grant sponsor: Bill & Melinda Gates Foundation (Grand Challenges in Global Health initiative) Contract grant sponsor: Shota Rustaveli National Science Foundation (Republic of Georgia) Contract grant number: DI/23/7-230/12 VC 2014 Wiley Periodicals, Inc. 88 Biopolymers Volume 103 / Number 2