Microarrays, in which several thousand individual parameters can be analysed simultaneously in biological samples using DNA, RNA, protein or antibody probes, have long become the international scientific standard. The same is true for next-generation sequencing methods with which a human genome can be analysed within a few days. The two techniques are often viewed as competing platforms. However, Dr. Günter Roth from the Centre for Biological Systems Analysis at the University of Freiburg is now daring to do something completely new. He is working on the development of a ‘microarray copier’ that can – almost at the push of a button – copy arrays of any type of molecule – DNA, RNA and proteins – from next-generation sequencing chips onto standard slides, thereby combining the world of microarrays with the sequencing world. The approach has a huge application potential for the production of detergent enzymes as well as vaccines.
All technological and biotechnological innovations have an important aspect in common, namely the miniaturisation of the basic idea. Microplates with 96 wells have long been replaced by biochips on which hundreds of thousands probes can be immobilised in separate spots in rows and columns. The basic idea has not changed much. “We use biochips for basically the same analyses microtitre plates were used for,” said Dr. Günther Roth from the Centre for Biological Systems Analysis (ZBSA) at the University of Freiburg. “The only difference is that the dimensions are a thousand to a million times smaller.”
Miniaturisation has considerably reduced the amount of sample and reagents required, even picolitre quantities (10-12 litres) are sufficient. On the one hand, this has the advantage that hundreds of measurements can be made with one droplet of blood. On the other hand, the system has the technical constraint that hundreds of thousands of tiny spots need to be immobilised on a small surface. And this can take time, even when a pipetting robot is used, and affect the quality of the molecules.
Although in-situ synthesis, i.e. production of the biomaterial directly on the substrate, is quicker than printing hundreds of thousands of spots, only relatively short molecules can be synthesised and a high level of impurities might be present. Using a different technique might be the sought-after solution: how about copying the molecules from the original, i.e. the genomic DNA of a cell, rather than synthesising them in a time-consuming process?
Günter Roth’s idea is a rather unconventional one, at least from a biological perspective: DNA, RNA and proteins are not synthesised de novo, but copied onto a slide. Roth is a systems biologist with two diploma degrees, one in biochemistry and one in physics, and is interested in biological as well as technical details. “Our ultimate goal is to develop a device that works like a Xeroxing machine for microarrays that is able to copy DNA into DNA, RNA or protein, simply by pressing a button.” Pure fiction? Not really. The first prototypes have been produced and Roth and his colleagues have already been able to copy DNA into DNA. “We can also copy DNA into proteins,” said Roth explaining that their microarray copier is based on the knowledge that dividing cells replicate their DNA, thereby creating two identical copies from one original DNA molecule, and that growing cells make an RNA copy of a DNA molecule and synthesise a protein from the information contained in an RNA molecule. While a Xeroxing machine works with cyan, magenta and yellow ink, Roth’s microarray copier works with enzyme mixtures for DNA, RNA and proteins. The Hans L. Merkle Foundation, which funds the development of high-potential and high-risk technologies and innovations, has also supported the development of the microarray copier.
The enzymes required for copying DNA, RNA and proteins have been known for 30 years. A mix of enzymes (e.g., DNA/RNA polymerases) and other reagents that cells need to produce DNA, RNA or proteins can be purchased as kits from many manufacturers. “The trick is to modify the established biological procedures in a way that ensures that a copy is made from the DNA and transferred to a different surface,” explained Roth who is head of the Microarray Copying group at the ZBSA. As everything starts with DNA, a DNA template is all that is required. One possibility of directly using DNA as a template is to use a next-generation sequencing chip which contains millions of small plastic beads. Roth and his colleagues use a 454 sequencing system which involves beads that are loaded into the wells of a picotitre plate in such a way that each well contains only one bead with a gene fragment that differs from those on the other beads.
“We had to find a way to get the DNA from the beads to the surface of a microscopic slide. We found that the best method was to use a mix of DNA enzymes and place a microscopic slide over the wells,” Roth explained. During PCR (polymerase chain reaction), the enzyme DNA polymerase starts synthesising new DNA from a primer attached to the microscopic slide. The PCR products are thus immobilised onto the slide surface. “The enzyme does not mind where the products end up,” said the researcher going on to add, “we can thus modify the surfaces so that the DNA is immobilised at a predetermined site.” In the case of a Xeroxing machine, the mix of enzymes would be the inks, the DNA of the sequencing chip would be the master and the specifically coated surface, the copying paper. “We can determine whether we want to immobilise the DNA on the slide, which is what we do when we want to create a microarray. We can also immobilise the DNA on the walls of the wells. We do this when we want to produce a DNA stock that can be used for the production of RNA and protein,” Roth said.
Copying DNA into a protein is more difficult than copying DNA into DNA. The researchers need to adjust the chemistry in order to prevent the product from floating around in the mixture, and instead bind to the slide surface as desired. The researchers are using well-tried components – His (histidine) affinity tags grafted onto proteins and nickel-NTA surfaces to which the His tags bind as copying paper – in their attempt to turn their vision into reality. The next idea is to create a shortcut – omission of the beads and direct use of genomic DNA. This has many advantages: no time-consuming and costly production of beads and easy handling. Without the beads, there is also more space in the wells and larger quantities of enzyme mixes can be added, resulting in even more efficient and quicker copying. Essentially, the imaginable uses are limitless. ”The microarray copier works like a Xeroxing machine,” repeated Roth, “it is up to the users themselves to decide what they want to copy.” Roth and his colleagues are currently working on proving the feasibility of the method for some potential applications. Roth has numerous ideas for application, including the use of the copier for identifying tumour markers, detecting antibodies in patients with allergies and autoimmune diseases and for optimising detergent enzymes. It also has the potential to reduce vaccine development to a few days. This can be achieved by copying pathogen proteins in microarrays to a surface and exposing them to patient antibodies which then bind to the proteins. The copied pathogen proteins serve as epitopes that can be used as active vaccines following the production of larger protein quantities with bacteria. Roth has already filed a patent for the process of discovering and producing vaccines. Roth himself is specifically focussed on antibodies for copied HPV proteins and has plans to study the regulatory network involving copied Reelin, an extracellular matrix glycoprotein that regulates neuronal migration and positioning processes. “It would be nice to see the little copier in every 5th laboratory or so and hear people say that it is quite handy to have,” said Roth highlighting his long-term vision.
Further information:Dr. Günter RothMicroarray CopyingZBSA (Centre for Biological Systems Analysis)University of FreiburgHabsburgerstr. 4979104 FreiburgTel.: +49 (0)761 / 203 - 97167Fax: +49 (0)761 / 203 - 5116E-mail: guenter.roth(at)zbsa.uni-freiburg.de