Research Challenge How does viral load change in response to various treatments? Exactly how efficient is a gene-therapy delivery vector? Which transgenic plants are optimal for cross germination? Answers to these questions require absolute quantification—comparing the PCR cycle thresholds of unknown targets to a standard PCR curve. Success with this method depends on having an accurate standard and an ability to link it to results with unknown sequences. Thus, the gnawing concern for scientists: Just how reliable are the results?

The 12.765 Digital Array. Click image for details.

The Solution: BioMark 12.765 Digital Arrays

At last, there's a practical way to "absolutely" quantify the number of target-sequences in a sample—the BioMark 12.765 Digital Array. This breakthrough is possible through integrated channels and valves that partition mixtures of sample and PCR reagents into 765 nano-volume reactions. This way, single copies of the target sequence can be isolated and detected with absolute reliability.

This approach relies on discovering the correct concentration of the analyte within a mixture. The scientist prepares five-fold or ten-fold dilutions of the sample and loads the various dilutions, together with reagents, in one of the twelve partitioning domains on the chip. If a domain shows too many positives—in the extreme, all 765 partitions register as positive—it's impossible to extrapolate the number of copies in each partition. If fewer than 10 partitions register as positive, the sampling error is too high. An appropriate dilution gives in the range of 30 to 200 positives, a good statistical sampling.

The chip handles 12 mixtures at once. After partitioning on the NanoFlex™ IFC controller, the mixtures are placed on the BioMark™ system, for thermal amplification and fluorescence detection. Our data proves that digital arrays give single-copy detection within each partition, a requirements for absolute quantification. See data.

The 12.765 Chip at a Glance

Parameter		Specification
Samples		12
Partitions per sample		765
Input volume per well		6 nanoliters
Liquid-transfer steps		12
Reactions per chip		9,180
Experiment tracking		Chip-specific barcodes
Chip setup and valve control		NanoFlex IFC Controller
Detection		BioMark System for Genetic Analysis

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