Rapid hybridization of nucleic acids using isotachophoresis
Moran Bercovici, Crystal M. Han, Joseph C. Liao, and Juan G. Santiago
- We create a 10 pL virtual reaction chamber in which we simultaneously concentrate, mix and accelerate nucleic acid hybridization.
- Demonstrated 10,000-fold acceleration in DNA hybridization compared to standard hybridization, reducing hybridization time from days into seconds.
- Closed-form analytical solution and validation experiments for reactions under ITP, revealing a new time scale for reactions.
Nucleic acid hybridization
Nucleic acid hybridization is ubiquitous in molecular biology, biotechnology, and biophysics, and has been instrumental in the development of numerous important techniques including genetic profiling, pathogen identification, sequencing reactions, and single-nucleotide polymorphism typing.
Several factors, namely diffusion, transport, and reaction rates limit hybridization at low concentrations. While diffusion and transport limitation can be effectively overcome by use of devices such as mixers and flow channels, reaction rates remain a major bottleneck toward achieving rapid hybridization of nucleic acids at low concentrations. Polymerase chain reaction is commonly employed to amplify signals. However, there is a growing need to find alternatives that could be simpler, faster and cheaper to implement. Amplification-free hybridization is particularly important for applications intended for use beyond the conventional lab settings, such as in point of care diagnostics, forensics, and environmental monitoring.
Rapid hybridization assay
Fig. 1. Schematic depicting acceleration of nucleic acid hybridization reactions using ITP. Two single-stranded DNA species A and B are focused at a narrow (order 10 μm) interface between the TE and LE in a microchannel. TE and LE are chosen such that their mobility bound all of the nucleic acid mobility. In this model system, species A is mixed with TE, and species B is mixed with LE, thus reaction occurs only at the interface where both species focus. The high concentrations of reactants at the interface lead to a corresponding increase in hybridization reaction rate. The arrow lengths at the top respectively denote the relative speed of species A in TE, of the ITP interface, and of species B in LE (LE and TE ions migrate at velocities equal to that of ITP interface).
Analytical model and validation experiments
Figure 3: (a) Analytical and numerical predictions vs. ITP-aided DNA hybridization data with no fitting parameters (We quantify forward kinetic reaction rate in independent experiments). Shown are normalized fluorescence signal over time. In the inset we show raw example experimental data at target concentrations of 1, 10, and 100 nM versus 10 nM concentration of molecular beacons (probe length 28-mer; stem length 7-mer). TE was 50 mM Tricine and 100 mM Bistris, and LE was 250 mM HCl, 500 mM Bistris, 2 mM MgCl2, 0.1 % PVP. In the main plot, we show the same data plotted in log-log scale with the time axis normalized by the characteristic time scale predicted by the model. (b) Experimental demonstration of the predicted 800 fold hybridization speed-up for O(10 nM) DNA oligonucleotides. Fraction hybridized plotted in log-log scale for ITP and standard hybridization (without ITP preconcentration) cases as a direct demonstration of rapid hybridization by the assay. The experiment was conducted using 10 nM concentration of molecular beacons and 50 nM target concentration. For ITP, we used TE of 30 mM Tricine and 60 mM Bistris, and the same LE used for Figure 3a. The standard hybridization data were obtained from pressure-driven flow system where reactants were premixed in the LE buffer used in the ITP experiments. The model prediction solid lines are plotted based on independent kinetic rate measurement, interface width, and ITP speed values extracted from the experiment.
10,000X acceleration demonstration
Fig. 4. Experimental demonstration of the 960-fold and 14,000-fold hybridization acceleration for reactions with order 10 nM and 100 pM DNA oligonucleotides, respectively. The fraction of reactants hybridized is presented against time for both standard hybridization (right two curves) and ITP-based hybridization (left two curves). Each data point shown for ITP-based hybridization is the average of four realizations with range bars representing the full absolute range of measured values. Solid lines denote theory predictions based on Eq. 3 using experimentally measured kon of 4750 M−1 s−1. For the case of 10 nM molecular beacons and 20 nM target concentration (squares), the half-times for standard and ITP-based hybridization were 2.4 h and 9 s, respectively, constituting a 960-fold hybridization speed up. For the case of 50 pM beacons and 500 pM target concentration (circles), we compare experimental data of ITP hybridization with the theory-prediction for the standard hybridization since standard hybridization experiment required more than 10 days to reach steady state. The expected half time of 3.7 days of the second order standard hybridization was significantly reduced to an experimentally measured 23 s using ITP, indicating a 14,000-fold hybridization speed up.
- Bercovici M. Han C.M., Santiago J.G. and Liao J.C., “Rapid DNA hybridization using isotachophoreis”, PNAS 109, 11127–11132 (2012).