Abstract
The demand for solutions to perform forensic DNA profiling outside of centralized laboratories is increasing. We here demonstrate highly sensitive STR amplification using a silicon micro-PCR (µPCR) chip. Exploiting industry-standard semiconductor manufacturing processes, a device was fabricated that features a small form factor thanks to an integrated heating element covering three parallel micro-reactors with a reaction volume of 0.5 µl each. Diluted reference DNA samples (1 ng–31 pg) were amplified on the µPCR chip using the forensically validated AmpFISTR Identifier Plus kit, followed by conventional capillary electrophoresis. Complete STR profiles were generated with input DNA quantities down to 62 pg. Occasional allelic dropouts were observed from 31 pg downward. On-chip STR profiles were compared with those of identical samples amplified using a conventional thermal cycler for direct comparison of amplification sensitivity in a forensic setting. The observed sensitivity was in line with kit specifications for both µPCR and conventional PCR. Finally, a rapid amplification protocol was developed. Complete STR profiles could be generated in less than 17 minutes from as little as 125 pg template DNA. Together, our results are an important step towards the development of commercial, mass-produced, relatively cheap, handheld devices for on-site testing in forensic DNA analysis.
Highlights
Forensic DNA profiling is performed almost exclusively by multiplex polymerase chain reaction (PCR) amplification of short tandem repeat (STR) regions[1,2]
The advantages of the proposed silicon chip-based solution to be incorporated into a Rapid DNA instrument are threefold: (i) integrated heaters and sub-μl reaction volumes allow for a small form factor; (ii) small PCR reactors etched into thermally conductive silicon, surrounded by thermal insulation trenches, enable rapid thermal cycling with limited power consumption; (iii) chip fabrication using industry-standard semiconductor manufacturing processes enable highly reproducible and relatively cheap mass production
This work compared the sensitivity of the forensic AmpFlSTR Identifiler Plus kit using a conventional thermal cycler and an integrated μPCR chip
Summary
Forensic DNA profiling is performed almost exclusively by multiplex polymerase chain reaction (PCR) amplification of short tandem repeat (STR) regions[1,2]. These repetitive sequences are highly polymorphic allowing for the generation of very distinctive profiles from minute amounts of DNA. Multiplex PCR amplification is an expensive, time-consuming and labor-intensive process requiring a dedicated laboratory setting with limited potential for on-site testing. A rapid amplification protocol was developed harnessing the full potential of on-chip amplification and allowing complete STR profiles to be generated in a fraction of the time normally required
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