Overview of the single-cell SD genotyping chip workflow.

<p>(<b>a</b>) The chip is composed of PDMS bonded to a microscope slide with a bonded glass coverslip over the array and surrounding oil channel to prevent sample evaporation. (<b>b</b>) Cell-PCR mix suspension is pipetted directly into the inlet reservoir flows into the wells of the array by applying vacuum to the outlet port. Once the sample volume is loaded into the wells, the main channel is flushed with oil to fully digitize each 8nL volume. The arrays are imaged to identify wells containing single (or more) cells. PCR is performed in the digitized volumes by thermalcycling of the entire chip, and fluorescence is quantified at endpoint in three fluorescence channels (FAM for the amplification probe, HEX for the mutant allele-specific probe and Cy5 for the wild-type-specific probe). In wells containing a single-cell and with amplification probe fluorescence above a set threshold, as well as allele-specific probe fluorescence above their respective thresholds, we can generate QC statistics for the array and determine zygosities of single cells.