An inexpensive plastic disk disposable was designed for digital polymerase chain reaction (PCR) applications with a microfluidic architecture that passively compartmentalizes a sample into 1000 nanoliter-sized wells by centrifugation. Well volumes of 33 nL were attained with a 16% volume coefficient of variation (CV). A rapid air thermocycler with aggregate real-time fluorescence detection was used, achieving PCR cycle times of 33 s and 94% PCR efficiency, with a melting curve to validate product specificity. A CCD camera acquired a fluorescent image of the disk following PCR, and the well intensity frequency distribution and Poisson distribution statistics were used to count the positive wells on the disk to determine the number of template molecules amplified. A 300 bp plasmid DNA product was amplified within the disk and analyzed in 50 min with 58-1000 wells containing plasmid template. Target concentrations measured by the spinning disk platform were 3 times less than that predicted by absorbance measurements. The spinning disk platform reduces disposable cost, instrument complexity, and thermocycling time compared to other current digital PCR platforms.

Two microchannel manufacturing methods—xurography of double-sided tape and glass etching (lithography and wet etching)—were compared using DNA melting analysis. A heterozygous mutation (3 base-pair deletion) was distinguished from wild type (normal) DNA in 10 nL (xurography and glass etching) and 1 nL (xurography) volumes. The results of the 10 nL and 1 nL melting curves were compared to results using commercial high-resolution instrumentation with 10 µL volumes. These 1000-fold and 10 000-fold volume reductions reduced the signal-to-noise ratio (SNR) only 29-fold and 40-fold for xurography (10 nL and 1 nL, respectively,) and 39-fold for 10 nL glass etched microchannels, still providing adequate discrimination for mutation detection. The reduced SNR of the glass etched microchannels compared to the tape microchannels was due to the in-house bonding process which gave poor optical quality on the surface of the microchip. Xurography of double-sided tape reduces the cost by 20 fold and is four times faster to manufacture than glass etching. Microchips created using the rapid prototyping technique of xurography are a reasonable prototyping alternative to channels created using traditional glass etching for DNA mutation detection.

Solution-phase, DNA melting analysis for heterozygote scanning and single nucleotide polymorphism (SNP) genotyping was performed in 10 nl volumes on a custom microchip. Human genomic DNA was PCR amplified in the presence of the saturating fluorescent dye, LCGreen Plus, and placed within microfluidic channels that were created between two glass slides. The microchip was heated at 0.1 degrees C/s with a Peltier device and viewed with an inverted fluorescence microscope modified for photomulitiplier tube detection. The melting data was normalized and the negative first derivative plotted against temperature. Mutation scanning for heterozygotes was easily performed by comparing the shape of the melting curve to homozygous standards. Genotyping of homozygotes by melting temperature (T(m)) required absolute temperature comparisons. Mutation scanning of ATM exon 17 and CFTR exon 10 identified single base change heterozygotes in 84 and 201 base-pair (bp) products, respectively. All genotypes at HFE C282Y were distinguished by simple melting analysis of a 40-bp fragment. Sequential analysis of the same sample on the gold-standard, commercial high-resolution melting instrument HR-1, followed by melting in a 10 nl reaction chamber, produced similar results. DNA melting analysis requires only minutes after PCR and is a simple method for genotyping and scanning that can be reduced to nanoliter volumes. Microscale systems for performing DNA melting reduce the reagents/DNA template required with a promise for high throughput analysis in a closed chamber without risk of contamination.

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