Multiplex STR amplification sensitivity in a silicon microchip

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.

Figure 1

Top view showing the complete chip including integrated heater, air trenches surrounding the reaction cavity, three inlets and outlets and the PC. The integrated heater and resistance temperature detector are wire-bonded to the 10-pin connector. The insertion shows a schematic overview of the reaction chambers, access holes, channels and air trenches etched in silicon. Indicated in purple are the front side channels created by a front side etch, the blue layers represent the channels created by front and backside etching of the silicon substrate, thereby piercing through the silicon substrate. Depth of the front side etch and backside etch is 0.25 mm and 0.17 mm respectively.

Figure 2

CE profiles generated after (A) Off-chip amplification and (B) On-chip amplification of a serial dilution of input material (1 ng–31 pg) and a no template control (NTC). All experiments were conducted in triplicates (n = 3).

Figure 3

(A) Percentage of observed alleles throughout the dilution series for both Off-chip (♦) and On-chip (●) amplifications. (B) Peak height ratio of all heterozygous loci throughout the dilution series for both Off-chip (♦) and On-chip (●) amplifications. All amplifications were conducted in triplicates (n = 3).

Figure 4

Comparison of CE profiles generated Off-chip (A) and On-chip (B) using identical input concentrations (0.1 ng/µl, 0.05 ng/µl, 0.025 ng/µl). All experiments were conducted in triplicates (n = 3).

Figure 5

CE profiles generated after rapid on-chip PCR amplification of a serial dilution of input material (1 ng–31 pg). All experiments were conducted in triplicates (n = 3).