Abstract
The demand to perform forensic DNA profiling outside of centralized laboratories and on the crime scene is increasing. Several criminal investigations would benefit tremendously from having DNA based information available in the first hours rather than days or weeks. However, due to the complexity and time-consuming nature of standard DNA fingerprinting methods, rapid and automated analyses are hard to achieve. We here demonstrate the implementation of an alternative DNA fingerprinting method in a single microchip. By combining PCR amplification and HyBeacon melting assays in a silicon Lab-on-a-chip (LoC), a significant step towards rapid on-site DNA fingerprinting is taken. The small form factor of a LoC reduces reagent consumption and increases portability. Additional miniaturization is achieved through an integrated heating element covering 24 parallel micro-reactors with a reaction volume of 0.14 µl each. The high level of parallelization allows the simultaneous analysis of 4 short tandem repeat (STR) loci and the amelogenin gender marker commonly included in forensic DNA analysis. A reference and crime scene sample can be analyzed simultaneously for direct comparison. Importantly, by using industry-standard semiconductor manufacturing processes, mass manufacturability can be guaranteed. Following assay design and optimization, complete 5-loci profiles could be robustly generated on-chip that are on par with those obtained using conventional benchtop real-time PCR thermal cyclers. Together, our results are an important step towards the development of commercial, mass-produced, portable devices for on-site testing in forensic DNA analysis.
Highlights
Human DNA fingerprinting is currently almost exclusively performed based on short tandem repeat (STR) region identification[1,2]
The gold standard method for generating STR profiles was optimized about two decades ago and consists of a combination of multiplex polymerase chain reaction (PCR) and a subsequent fragment sizing using capillary electrophoresis (CE)[5]
STR profiling using CE-based size separation has been the gold standard for traditional DNA fingerprinting analysis, attempts at miniaturization of CE by on-chip implementation have been unable to deliver the single-basepair resolution that is required for forensic applications
Summary
Human DNA fingerprinting is currently almost exclusively performed based on short tandem repeat (STR) region identification[1,2]. The PCR LoC based solutions described in the literature are mainly focused towards point of care applications and are limited in terms of reaction multiplexing[13,14] Many of these LoCs rely on-chip reagent storage, which is challenging and results in an increased device fabrication complexity[15,16]. In this paper a forensic LoC was designed, fabricated and tested capable of performing multiple PCR amplifications and STR detection without the need to perform CE analysis on-chip. Temperature monitoring was accomplished by a resistance temperature detector (RTD) deposited directly on the silicon surface The performance of this novel LoC to generate STR profiles using HyBeacon technology was evaluated. Complete STR profiles of three reference samples were generated on-chip and compared to off-chip results
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