First characterization of a biphasic, switch-like DNA amplification

Abstract

We report the first DNA amplification chemistry with switch-like characteristics: the chemistry is biphasic, with an expected initial phase followed by an unprecedented high gain burst of product oligonucleotide in a second phase. The first and second phases are separated by a temporary plateau, with the second phase producing 10 to 100 times more product than the first. The reaction is initiated when an oligonucleotide binds and opens a palindromic looped DNA template with two binding domains. Upon loop opening, the oligonucleotide trigger is rapidly amplified through cyclic extension and nicking of the bound trigger. Loop opening and DNA association drive the amplification reaction, such that reaction acceleration in the second phase is correlated with DNA association thermodynamics. Without a palindromic sequence, the chemistry resembles the exponential amplification reaction (EXPAR). EXPAR terminates at the initial plateau, revealing a previously unknown phenomenon that causes early reaction cessation in this popular oligonucleotide amplification reaction. Here we present two distinct types of this biphasic reaction chemistry and propose dominant reaction pathways for each type based on thermodynamic arguments. These reactions create an endogenous switch-like output that reacts to approximately 1 pM oligonucleotide trigger. The chemistry is isothermal and can be adapted to respond to a broad range of input target molecules such as proteins, genomic bacterial DNA, viral DNA, and microRNA. This rapid DNA amplification reaction could potentially impact a variety of disciplines such as synthetic biology, biosensors, DNA computing, and clinical diagnostics.

A mathematical model for a biphasic DNA amplification reaction

“…Using reasonable parameter estimates based on values reported in the literature, we found that this model explains all key characteristics of the biphasic reaction described in [11]. Our model reproduces two distinct DNA amplification periods and two periods of stalled growth as observed in UDAR trials.…”

“…The final parameters were chosen to fit the reaction initiation observed in experimental data for the template studied in this work while keeping reasonable values for the DNA association kinetics. The free energy of the trigger association to the looped template, given by the difference in free energy between the 3 0 toehold association with the trigger and the loop secondary structure, was one parameter shown in [11] to correlate with the inflection point of the first phase. This correlation is consistent across the biphasic amplification reactions originally reported.…”

“…Recently, we constructed an isothermal DNA amplifying template where the complementary binding regions contain a palindromic sequence. This construction produces intriguing biphasic behaviour [11] (figure 1). The pair of palindromic sequences cause the templates to fold into an energetically favourable hairpin structure which remains closed in the absence of trigger.…”

“…Since this second phase greatly increases output signal, it is important to understand the factors that cause amplification to resume in this biphasic reaction. The goal of this work is to first mathematically model key reaction mechanisms suggested in [11] that may explain each phase of this reaction and then to simulate the mechanisms with reasonable approximations for each kinetic component. We compare this model to experimental data produced from an ultrasensitive DNA amplification reaction (UDAR) template and show that without the key reaction mechanisms, the model deviates from the data.…”

“…Biphasic DNA amplification output: representative outputs of the biphasic DNA amplification reaction UDAR. Type I and II templates can be distinguished by the free energy of DNA association, which is described in detail in [11]. Both types of templates exhibit two growth phases, but in type I templates, the first growth phase is slower than the second growth phase and the first plateau is relatively long.…”