Researchers at Germany's University of Freiburg have developed a method to perform highly parallel PCR amplification in a picowell array while simultaneously immobilizing the PCR products onto a microscope slide for future analysis.
In a paper published in June in Lab on a Chip, the researchers demonstrated the approach — called picowell array solid-phase PCR, or PWA-SP-PCR — using a 100,000-well array of 19 pL reaction volumes. They showed that it could efficiently amplify and immobilize DNA templates of up to 1,500 base pairs, as well as single DNA molecules for digital PCR.
Günter Roth, head of the lab-on-a-chip assays group at the Department of Microsystems Engineering at the University of Freiburg, told BioArray News that the ultimate aim of the group is to develop a "microarray copier" that can copy arrays of any type of molecule — DNA, RNA, proteins, or even chemical compounds — onto standard slides.
The Lab on a Chip paper serves as proof of concept of the method's ability to serve as a DNA copier, he said. Next, the group is planning to adjust the chemistry to create a protein copier and plans to follow that up with a version that copies RNA. "We want to show that this is like a Xeroxing machine for microarrays," he said.
Since DNA applications have the most short-term promise, the group is currently focused on "integrating a standard sequencing run with our copying technology," he said. Potential partners could include any next-generation sequencing company, but he noted that the chips used for the Roche GS FLX and Life Technologies' Ion Torrent are best suited to the approach. Roth said the group is in discussions with several potential commercial partners and that "we welcome them all at the moment."
The picowell array described in the paper includes 100,000 wells that hold DNA sequences along with liquid-phase and solid-phase PCR primers. The array is covered with a microscopic slide and then PCR is carried out — liquid-phase until those primers are depleted, and then solid-phase, which immobilizes the products onto the slide surface. The copies are located at the exact same position of the original DNA sequence and the slides can be used for subsequent analysis without any additional equipment or transfer steps.
Roth and colleagues explain in their Lab on a Chip paper that this approach improves upon emulsion PCR, which requires more time for annealing and extension, during which single droplets can merge to create chimeric DNA sequences.
Roth told BioArray News that PWA-SP-PCR could potentially replace emulsion PCR in next-gen sequencing workflows, but stressed that it would "have to be tested."
He noted that the method could be used to make whole-genome arrays or aptamer arrays just by adjusting the template DNA. In the case of protein arrays, the first PCR step would be modified to create expression-ready DNA, and then the copying step would use a cell-free expression mix to create proteins on the microscope slide.
Current protein arrays, such as the ProtoArray from Life Technologies, are on the order of around 9,000 proteins per chip, so the ability to copy 100,000 proteins onto a slide could be quite an advance, though Roth noted that the protein work is still in the early stages.
The RNA work is even farther off, he said, "because there is no real application. If you tell people we can make an RNA microarray with 100,000 different RNAs then they have to think about how they can use it because nobody can produce that at the moment."
Because RNA degrades quickly it's not possible to manufacture an array and ship it to a customer, he said, but if the researcher instead receives the picowell "master" with template DNA, he or she could copy the RNAs onto a slide when it's time to do an experiment.
This model is a departure from current methods that make microarrays "spot by spot," he said. Instead, "we can take all these natural enzymes and use them to copy molecules. We are the copying people."