Pulse-controlled amplification

Similar to PCR, pulse-controlled amplification (PCA) relies on the exponential amplification of a specific nucleic acid target fragment for subsequent detection. Amplification is achieved through the binding of target-specific, complementary oligonucleotide primers to template DNA, followed by primer extension by a DNA polymerase enzyme. PCA also relies on thermal cycling, however, instead of time-consuming alternating heating and cooling of the total reaction volume (“global heating”), rapid sub-millisecond voltage pulses are applied to an array of 75 gold-coated tungsten wires (15 μm diameter, 200 nm Au coating), causing ultra-fast heating within only a micrometer-sized liquid layer surrounding each wire (“local heating”). The remaining bulk of the reaction volume (more than 99%) is kept at the base temperature used for annealing and elongation. The approach of “local heating” denatures double stranded (ds) DNA only within the heated layer surrounding the wires, which makes it necessary for part of the reaction to be localized as well. This is achieved by attaching one of the primers to the micro-scale conductive metal structures (in this study gold-coated tungsten wires). The other primer remains free in solution, providing the kinetic advantages of a free reaction. As a result, the dsDNA denaturation step of the amplification reaction requires only a fraction of the energy usually required to thermocycle the total reaction volume. Local heating allows the wires to cool off after the voltage pulse that drives the denaturation step by thermal diffusion on a millisecond time scale. The bulk of the reaction volume serves as cooling reservoir for an entirely passive cooling process of the embedded wires, resulting in ultra-fast thermal cycles. This reduces the total time of the amplification process by a factor of up to ten compared to PCR, as hundreds of energy pulses can take place in a short amount of time. Like qPCR, amplification can be traced in real time using intercalating dyes or, as in our study, hydrolysis probes (Fig 1).

Schematic of the PCA process and the interaction of primers and gold coated wires (A) (reduced) Thiol-modification was added to the 5’-end of the primer using a poly-A-tail and Int Spacer 9, allowing for a strong AU-S bond and immobilization of the primer to the gold-coated wires (B) Annealing, probe hybridization and Elongation (C) PCA uses thermal cycling but instead of time-consuming alternating heating and cooling of the whole reaction mixture, rapid energy pulses are applied to the gold wires, causing ultra-fast local heating and dsDNA denaturation.

Currently, PCA is performed on a prototype instrument, the Pharos Micro (GNA Biosolutions, Martinsried, Germany) utilizing prototype disposable chips, which contain the amplification reactions (GNA Biosolutions, Martinsried, Germany). To optimize assays, different parameters of the run are adjustable, including base and lid temperature of the Pharos Micro [°C], heating time [μs], cycle time [s], number of cycles, and thermalizing time [s]. For primer design, the guidelines in Table 1 should be followed. For successful PCA, it is critical to avoid primer dimers when designing primers, especially for the thiolated-primer used for functionalization of the wires.