Fluidigm miniaturises the time and cost of PCR genotyping

By Dr Matt Wilkinson

- Last updated on GMT

Related tags Polymerase chain reaction

US-microfluidics expert Fluidigm has launched a new dynamic array
for its BioMark instrument that increases the efficiency of PCR
(polymerase chain reaction) genotyping while reducing the amount of
costly equipment and reagents needed.

By using recently developed microfluidic devices known as integrated fluid circuits (IFCs), Fluidigm has managed to reduce the 'logistical friction' of associated with running large genotyping experiments. Traditional approaches to large scale genotyping involve numerous microwell plates, large amounts of costly reagents and dispensing robotics to cope with the intensive liquid handling. According to Mike Lucero, executive vice president of Sales and Marketing at Fluidigm, the IFCs are three dimensional microfluidic devices that have 48 reagent ports and 48 reaction ports that allow 2,304 parallel nano-volume reactions in each IFC. This is achieved by driving a precise amount of reagents and reactants into chambers in the IFC that are limited by valves. "Microfluidics got the reputation for not being able to find the appropriate application - but its inside out because in the past microfluidics didn't have the valves so they couldn't achieve the integration and miniaturisation that we have now - it's the valves that make the difference,"​ said Lucero The valves are integrated into the chips with each IFC containing about 7,500 silicone rubber valves that are controlled by applying pressure to certain 'control channels' in the chip. "This is a really important product and the type of products we're making are very similar to integrated circuits -, these are essentially fluid circuits with the valves being analogous to the logic gates,"​ said Lucero. When the valves open the fluids are pushed into the reaction wells where the reagents and reactants mix to allow quantitative PCR genotyping reactions. "You can do 48 individual reactions for each 48 samples and this gives you six times more data than you would get with a 384-well plate system,"​ said Lucero. The new BioMark 48.48 dynamic arrays allow researchers to use their current reagents of choice - just a lot less of them. The dynamic arrays not only produce more data than traditional systems, they also reduce the number of pipetting steps required - only 96 pipetting steps are needed to conduct 2,304 reactions. Nevertheless, the chip is designed to be compatible with standard SBS (Society of Biomolecular Screening) pipetting robots to further increase reaction efficiency. "With our device we reduce the amount of Taq polymerase needed by 50 times and there are laboratories that spend hundreds of thousands of dollars on Taq polymerase in three months - that means that rather than spending $100,000 on reagents they only need to spend $2,000,"​ said Lucero. The BioMark system was initially aimed at real-time PCR applications such as gene expression analysis using fluorescent reporter probes that measure the amount of a specific sequence in real-time. "What you're looking for is the time when the amplification reaction crosses a certain threshold, that requires a certain amount of primers and probes to do it, but it's not sensitive to complete mixing,"​ said Lucero. The new application is a little more complicated as the amounts of reagent need to be precisely controlled and complete mixing is crucial. This is why the new chip architecture was necessary as it efficiently mixes the reagents in the reaction wells. "Genotyping is not a real time application, end point PCR depends on the primer and probe concentrations at the start of the reaction, and you use the [fluorescent signal] intensity at the end of the reaction, which is completely dependent on the primers and probes, to do the cluster analysis,"​ said Lucero.​ The company has also developed a digital PCR array that was launched in December last year. Digital PCR is the method of choice to achieve absolute quantification but has been too impractical for routine use until now. The technique works on the principal that target sequences can be counted if a sample is diluted to such an extent that only one target molecule may be present in a well after the sample is distributed to hundreds of reaction wells. "It's a ridiculously tedious experiment to do, but people do it when they need to know absolutely how much is in a sample,"​ said Lucero. The digital array brings this laborious reaction into the hands of laboratory workers by eliminating the need for thousands of pipetting steps and increasing the accuracy of the reactions. "One of the most important applications is to be able to detect a mutant genome in a large background of normal genomes - and this could be used to develop tests for a specific cancerous genome in the blood,"​ said Lucero.

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