From Quantitative PCR to Digital PCR
SNP detection will tell you if a specific base-pair is present or not. But it will not reveal how much of a certain DNA (e.g. HIV virus DNA) is present in a sample. That is done by qPCR (quantitative PCR), using fluorescent markers. These markers will result in an optical signal, a signal that gets stronger as the DNA is multiplied. By timing precisely when the light intensity crosses a threshold, it is possible to determine how much DNA was present to begin with.
qPCR is a robust and well established technique. It can be implemented with a comparable silicon microfluidics platform than the one used for the SNP chip. And compared to commercially available qPCR tools, the efficiency of a chip-solution proves equivalent. However, the technique has some drawbacks that make it a lengthy and cumbersome procedure, even if carried out on a lab-on-chip. The most important is the need to compare the light intensity with accurate calibration curves, curves that have to be made through additional, very precise experiments.
An alternative is to use digital droplet PCR (ddPCR). The idea is to divide the sample into many small droplets, so that each droplet contains none to at most a few units of the target DNA sequence. Then a PCR is done on all droplets, so that it becomes possible to see which droplets have target DNA and which don’t. By using the ratio of positive droplets to the total number of droplets, and applying Poisson statistics, the DNA concentration in the original volume can be calculated.
To implement ddPCR, imec has collaborated with Panasonic and Janssen Pharmaceuticals to extend its microfluidic toolbox with a uniform droplet generator. This droplet generator is formed by a T-shaped channel that injects the PCR sample with fluorinated oil mixed with appropriate surfactants. The channels currently-used are 30 micrometer wide resulting in droplets of 50 to 200pL. These droplets then stream further and are collected in the S-shaped PCR reactor, which occupies a 3x3mm area. The optical fluorescence read-out is done using a CCD camera coupled to a microscope. The resulting fluorescent image is then analyzed to count the number of droplets and their fluorescence intensity.
With ddPCR, it is thus possible to measure the quantity of a DNA sequence in a sample without having to use calibration curves. And also in contrast to qPCR, it can be used to detect two DNA variants even if one is much less available than the other. In this type of multiplexed PCR, two fluorescent colors will be used. If the droplet size and DNA dilution is chosen well, there will be a number of droplets in which the rare variant shines out.
In the above designs, the heart of the PCR tool is formed by a microfluidic chip. But this chip still needs external heaters to drive the thermal cycles, pumps and valves to inject the sample in the chip, and readout tools (e.g. camera and microscope).
Imec’s bioelectronics specialists are now working on integrating these elements on the chip, developing a veritable, stand-alone PCR chip which could be used alongside other tools in microsized integrated lab equipment. A chip also that can be fabricated in a CMOS-compatible process flow, allowing mass fabrication of highly identical precision tools. Our first test runs with integrated aluminum heaters show great potential. And to replace the pumps, we are experimenting with designs in which the sample moves through the channels through capillary forces only. Equally important is integrating more of the DNA analysis flow in the chip. This includes, on the one end, DNA extraction from biological material, and, on the other end, the results readout and analysis.
PCR on chip allows a much faster analysis than with larger, desktop-sized tools. An analysis also that automates a number of error-prone interventions by lab technicians. And with the promise of mass-fabrication, such tools can be brought to the point of care, as part of the infrastructure needed for a more personalized and efficient diagnosis and therapy.
Source: MDT Magazine