Although DNA microarrays are just beginning to be investigated for diagnostics, they are becoming key tools for preclinical and clinical research. Gene-expression analysis continues to provide information on what is occurring on a molecular level and is crucial to discovering new drug candidates. According to a Frost and Sullivan report, “U.S. DNA Microarray Markets,” the total U.S. revenue was $446 million in 2005, expected to increase to $532 million by 2012.
A number of researchers provided up-to-date reports on DNA microarrays at Select Biosciences’ “Advances in Microarray Technology” conference held last month in Edinburgh.
One of the biggest issues with microarrays has been high cost and difficulties in making them. Febit Biotech’s (www.febit.de) Geniom® platform designs, synthesizes, applies, and analyzes microarrays. Customers can use company-provided designs or design their own. “We want to overcome the hurdle of so few microarrays and so many genes. Our instrument allows people to make and test as many microarrays as there are genes and organisms,” said Peer Staehler, CSO.
The platform utilizes microfluidic biochips that enable several assays: dynamic detection of hybridization and amplification, PCR-on-Chip, longmer synthesis, and signal-enhancing protocols. Most microarrays are analyzed via a confocal scanner with one image. However, dynamic detection offers the ability to take images at any time during the experiment and enables a melting-curve profile. The microfluidic system prevents the liquid from evaporating.
“This gives you a binding profile on the whole chip, on thousands of features, which corresponds to a melting-curve plot. Instead of creating one layer of information, you create 20 or 30 data sets,” added Staehler. This provides benefits such as in SNP-typing, distinguishing a mutant from a wild-type, and for optimization of oligos, quickly identify their binding behavior.
Another feature, Enzyme-on-Chip, enables reverse synthesis of DNA oligos in the biochip. This provides new applications like PCR-on-Chip or primer extension reactions. “We try to fuse the advantage of the microarray being a multiplex analysis with the high data quality of the PCR reaction,” explained Staehler.
More researchers are investigating miRNAs, which appear to be responsible for regulating gene expression. Kreatech Biotechnology (www/kreatech.com) has developed Universal Linkage System (ULS™) to label small RNAs. ULS contains a platinum complex that forms a bond with the N7 position of guanine nucleotide base. There are currently two kits available, the ULS Small RNA labeling kit and the miRACULS miRNA kit.
“When we developed the first two kits, we found out that customers wanted kits for smaller RNA amounts,” said Erik Jan Klok, Ph.D., product development manager, microarrays. “These can handle about one microgram of small RNA, but the new kit can handle about 300 nanograms. It is critical to use less material.”
The ULS method directly labels nucleotides independent of their size. This differs from enzymatic methods, which use linkers or adapters to label the small amount of molecules. As it uses a chemical to label, it is a 15–30 minute reaction, followed by a spin column (KREApure™) to remove dye molecules that haven’t reacted. “Since there are only two components in the reaction, there is easy control over the degree of labeling,” added Dr. Klok. In addition, there isn’t any modification of the original miRNA used for hybridization, which avoids any bias. .
There have been many factors preventing the use of diagnostic microarrays in the clinic. Bertrand Jordan, Ph.D., a consultant for the Marseille-Genopole project, discussed why it is not catching on like many thought it would. Originally there was a lot of discussion about expression profiles predictive or prognostic of various types of cancer, Dr. Jordan said. However, the use of microarrays to identify mutations and copy-number variation is an easier application.
“With microarrays, you can look at many mutations simultaneously and see whether they are present. Usually the clinical importance of the mutation is already established,” Dr. Jordan added. Many FDA-approved microarray diagnostics are of this type, like the Affymetrix(www.affymetrix.com) AmpliChip® CYP450 test, which indicates whether a patient has a version of a given gene.
Arrays that look at expression profiles are more technically difficult because one is looking at hundreds of genes and trying to measure the expression of each one in a tumor sample over a wide dynamic range.
“There is the issue of what the profile actually means and how well you have proven that a profile correlates with a good prognosis or a bad prognosis,” said Dr. Jordan. He explained that there have been many findings not substantiated in further studies because of many possible pit falls. “If the correlation between a profile and prognostic information is good and scientifically valid, it is not necessarily clinically useful. If it is not used to help make clinical decisions, there is no point in paying for expensive tests.”