Specific versus Nonspecific Isothermal DNA Amplification through Thermophilic Polymerase and Nicking Enzyme Activities

Abstract

Rapid isothermal nucleic acid amplification technologies can enable diagnosis of human pathogens and genetic variations in a simple, inexpensive, user-friendly format. The isothermal exponential amplification reaction (EXPAR) efficiently amplifies short oligonucleotides called triggers in less than 10 min by means of thermostable polymerase and nicking endonuclease activities. We recently demonstrated that this reaction can be coupled with upstream generation of trigger oligonucleotides from a genomic target sequence, and with downstream visual detection using DNA-functionalized gold nanospheres. The utility of EXPAR in clinical diagnostics is, however, limited by a nonspecific background amplification phenomenon, which is further investigated in this report. We found that nonspecific background amplification includes an early phase and a late phase. Observations related to late phase background amplification are in general agreement with literature reports of ab initio DNA synthesis. Early phase background amplification, which limits the sensitivity of EXPAR, differs however from previous reports of nonspecific DNA synthesis. It is observable in the presence of single-stranded oligonucleotides following the EXPAR template design rules and generates the trigger sequence expected for the EXPAR template present in the reaction. It appears to require interaction between the DNA polymerase and the single-stranded EXPAR template. Early phase background amplification can be suppressed or eliminated by physically separating the template and polymerase until the final reaction temperature has been reached, thereby enabling detection of attomolar starting trigger concentrations.

First Characterization of a Biphasic, Switch-like DNA Amplification

“…DNA amplification output is correlated to fluorescence, which increases and plateaus at approximately the same level as previously reported optimized EXPAR reactions 32,33 (dotted lines). Biphasic DNA amplification output is shown in solid lines.…”

“…The traditional EXPAR template (Figure 3 A,D) did not enter the second phase, even when initial trigger concentration (10µM) was above the plateau concentration of this template (5.58µM, Table SI 3). Inflection points of the traditional template were linearly correlated with the log 10 of the original trigger concentration as expected 33 . Inflection points in the first phase for both the type I and II looped templates were also linearly correlated with the log 10 of the original trigger concentration, but the correlation appeared slightly nonlinear during the second phase (Figure 3 E,F).…”

“…We hypothesize that the plateau is due to noncanonical behavior of the nickase enzyme that leaves a long unextendable trigger, as the nickase is operating in suboptimal conditions when compared to the polymerase 33 . It is also possible that a fully elongated trigger poisons the template; however, the mechanism behind the template poisoning is beyond the scope of this study.…”

Microscopic agents programmed by DNA circuits

“…This phenomenon is again consistent with the behaviour reported for solution-phase amplification systems: autocatalytic templates self-start after some time because the first-order PFL network enforced by the dual repeat template is intrinsically monostable. 24 In order to stabilize the "OFF" state (corresponding to a nonproductive state) we use a species-specific deactivation template (pseudo-template, pT) that catalyzes the addition of a short 3' tail to the trigger of the associated template ( Fig. 2e and Fig.…”

Polymerase/nicking enzyme synergetic isothermal quadratic DNA machine and its application for one-step amplified biosensing of lead (II) ions at femtomole level and DNA methyltransferase

“…Nevertheless, the EXPAR-based methods involve the weaknesses of early phase non-specific background amplification resulting from the binding of the polymerase to the single-stranded template. 14 Conversely, the linear amplification strategies such as RCA, SDA and NESA are much simpler than EXPAR and out of the non-specific background amplification. Though well established and widely applied, these linear amplification strategies still face challenges in the detection of trace targets because of their relative low sensitivities.…”