Sequence dependence of isothermal DNA amplification via EXPAR

Abstract

Isothermal nucleic acid amplification is becoming increasingly important for molecular diagnostics. Therefore, new computational tools are needed to facilitate assay design. In the isothermal EXPonential Amplification Reaction (EXPAR), template sequences with similar thermodynamic characteristics perform very differently. To understand what causes this variability, we characterized the performance of 384 template sequences, and used this data to develop two computational methods to predict EXPAR template performance based on sequence: a position weight matrix approach with support vector machine classifier, and RELIEF attribute evaluation with Naïve Bayes classification. The methods identified well and poorly performing EXPAR templates with 67–70% sensitivity and 77–80% specificity. We combined these methods into a computational tool that can accelerate new assay design by ruling out likely poor performers. Furthermore, our data suggest that variability in template performance is linked to specific sequence motifs. Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates. Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers. Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions.

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.…”

“…This fluorescence variation does not affect the shape and inflection points of the graph, however. Traces are shown for three different template types: a traditional EXPAR linear template (EXPAR1 32 ), a type I template (LS2 lowtG), and a type II template (LS3), respectively. The traditional EXPAR template was chosen from 384 published sequences as it had the highest separation between positive and negative controls.…”

Computer-assisted design for scaling up systems based on DNA reaction networks

“…While the value of some parameters, such as the release slowdown owing to coaxial stacking, can be hard to predict, the actual value can be measured through some basic experiments and then updated in DACCAD, allowing the quick correction of a design. Such parameters can also be slightly modified from their actual experimental value to encompass more specific or sequence-dependent effects [54,55]. Finding a way to fit such experimental parameters directly from fluorescence data could be an important future application.…”

Exponential amplification of DNA with very low background using graphene oxide and single-stranded binding protein to suppress non-specific amplification

“…The amplified trigger further captures the templates that are absorbed to the GO sheets; thus, the duplex released from the GO surface yields amplification cycle 1 in Fig. 1, leading to exponential amplification of the trigger [11,17].…”

“…The sensitivity of EXPAR should be very high, but it is reported to range from 1 pM to 10 aM due to a high background signal [11,[13][14][15][16]. Factors affecting EXPAR variability have been investigated extensively, but its development is hampered by the high background, even under optimal conditions with a trained experimenter [11,14,17].…”