On-chip laser-induced DNA dehybridization.

Detection of pathogens and relevant genetic markers using their nucleic acid signatures is extremely common due to the inherent specificity genomic sequences provide. One approach for assaying a sample simultaneously for many different targets is the DNA microarray, which consists of several million short nucleic acid sequences (probes) bound to an inexpensive transparent substrate. Typically, complex samples hybridize to the microarray and the pattern of fluorescing probes on the microarray's surface identifies the detected targets. In the case of evolving or newly emergent organisms, a hybridization pattern can occur that differs from any previously known sources. When this happens it can be useful to recover the hybridized DNA from the binding locations of interest for sequencing. Here we present the novel utilization of a focused Infrared (IR) laser to heat user-selected spots on the DNA microarray surface, causing only localized dehybridization and recovery of the desired DNA into an elution buffer where it is available for subsequent amplification or sequencing. The introduction of a focused dehybridization method for spots of interest suppresses the amount of background DNA to be analyzed from downstream processes, and should reduce subsequent sequence assembly errors. This technique could also be applied to high-density protein microarrays where the desire to locally heat spots for release of bound molecules is desired.

Under-three minute PCR: probing the limits of fast amplification.

Nucleic acid amplification is enormously useful to the biotechnology and clinical diagnostic communities; however, to date point-of-use PCR has been hindered by thermal cycling architectures and protocols that do not allow for near-instantaneous results. In this work we demonstrate PCR amplification of synthetic SARS respiratory pathogenic targets and bacterial genomic DNA in less than three minutes in a hardware configuration utilizing convenient sample loading and disposal. Instead of sample miniaturization techniques, near-instantaneous heating and cooling of 5 µL reaction volumes is enabled by convective heat transfer of a thermal fluid through porous media combined with an integrated electrical heater. This method of rapid heat transfer has enabled 30 cycles of PCR amplification to be completed in as little as two minutes and eighteen seconds. Surprisingly, multiple enzymes have been shown to work at these breakthrough speeds on our system. A tool for measuring enzyme kinetics now exists and can allow polymerase optimization through directed evolution studies. Pairing this instrument technology with modified polymerases should result in a new paradigm for high-throughput, ultra-fast PCR and will hopefully improve our ability to quickly respond to the next viral pandemic.