Two new research developments from the USA are bringing the dream of personalised medicine a step nearer. In the first, a team of researchers with the US Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab) has invented a technique in which DNA or RNA assays can be read and evaluated without the need of elaborate chemical labelling or sophisticated instrumentation.
Based on electrostatic repulsion - in which objects with the same electrical charge repel one another - the technique is relatively simple and inexpensive to implement, and can be carried out in a matter of minutes.
"One of the most amazing things about our electrostatic detection method is that it requires nothing more than the naked eye to read out results that currently require chemical labelling and confocal laser scanners," said Jay Groves, a chemist who led this research. "We believe this technique could revolutionise the use of DNA microarrays for both research and diagnostics."
Groves and members of his research group Nathan Clack and Khalid Salaita have published a paper on their technique in the journal Nature Biotechnology which is now available online. The paper is entitled Electrostatic readout of DNA microarrays with charged microspheres.
In their paper, Groves, Clack, and Salaita describe how dispersing a fluid containing thousands of electrically-charged microscopic beads or spheres made of silica (glass) across the surface of a DNA microarray and then observing the Brownian motion of the spheres provides measurements of the electrical charges of the DNA molecules. These measurements can in turn be used to interrogate millions of DNA sequences at a time. What's more, these measurements can be observed and recorded with a simple hand-held imaging device - even a cell phone camera will do.
"The assumption has been that no detection technique could be more sensitive than fluorescent labelling, but this is completely untrue, as our results have plainly demonstrated," said Groves. "We've shown that changes in surface charge density as a result of specific DNA hybridisation can be detected and quantified with 50-picometer sensitivity, single base-pair mismatch selectivity, and in the presence of complex backgrounds. Furthermore, our electrostatic detection technique should render DNA and RNA microarrays sufficiently cost effective for broad world-health applications, as well as research."
Your susceptibility to a given disease and how your body will respond to drugs or other interventions is unique to your genetic makeup. Under a personalised medicine plan, treatment effectiveness is maximised and risks are minimised by tailoring disease treatments specifically to you. This requires the precise diagnostic tests and targeted therapies that can stem from assays using a DNA microarray - a thumbnail-sized substrate containing thousands of microscopic spots of oligonucleotides (stretches of DNA about 20 base pairs in length) laid out in a grid.
Often referred to as 'gene chips', DNA microarray assays and their RNA counterparts have become one of the most powerful tools for gene-expression profiling, the identification of mutations, and the detection of multiple pathogens in patients afflicted either by multiple diseases or drug-resistant strains of diseases. Aside from their potential future role in personalised medicine, the widespread use of DNA microarray assay devices could have an immediate and profound impact on the treatment of diseases today.
In a typical experiment, a microarray is prepared and mounted in a well chamber and the DNA is hybridised (a standard technique in which complementary single strands of DNA bind to form double-stranded DNA 'hybrids'). A suspension of negatively-charged silica microspheres is then dispersed through gravitational sedimentation over the microarray surface, a process which takes about 20 minutes.
Because the substrate or background surface of the microassay is positively charged, the silica microspheres will spread across the entire surface and adhere to it. However, on surface areas containing double-stranded DNA, which is highly negatively charged, and on areas containing single-stranded DNA, also negatively charged but to a lesser degree than double-stranded DNA, the microspheres will levitate above the substrate surface, stacking up in "equilibrium heights" that are dictated by a balance between gravitational and electrostatic forces.
These electrostatic interactions on the microarray surface result in charge-density contrasts that are readily observed. Surface areas containing DNA segments take on a frosted or translucent appearance, and can be correlated to specific hybridisations that reveal the presence of genes, mutations and pathogens.
"Our technique is essentially a millionfold parallel version of the classic experiment used by Robert Millikan almost 100 years ago, when he determined the charge of a single electron by observing the positions of oil droplets levitated above a charged plate," said Groves.
There are a number of short-term 'next steps" for this research, Groves said, including testing its application in high-density arrays and pushing its resolution limits.
"Since the resolution of electrostatic-based imaging is determined by the number of particle-observations rather than by the diffraction limit of light, our readouts could serve as a form of ultramicroscopy," he said. "The real grand challenge for this technology, however, will be for us to find suitable industrial partners with whom we can work to see that useful new products actually make it to market."
Meanwhile, researchers at Temple University's School of Pharmacy in Philadelphia, US, are using DNA to customise prescriptions in order to prevent adverse drug reactions before a single dose is taken (Fig.1).
According to the US Department of Health and Human Services, more than 770000 people are killed or injured each year in the US by such reactions. At the top of the list of problem drugs is Warfarin, the most widely prescribed anticoagulant. That is why Evgeny Krynetskiy, associate professor and director of the Jayne Haines Center for Pharmacogenomics and Drug Safety, has focused research on that drug. "Prescribing this medicine is like trial and error in finding the right dosage that works best for you," says Krynetskiy. "Five milligrams is a typical dose, but a little less or a little more could have dramatic consequences or no benefit at all."
Doctors call this optimal dosage the therapeutic window, and Krynetskiy is trying to find it through pharmacogenomics, the study of a person's response to drugs based on their genetic makeup. It's a collaboration that crosses campuses and includes Krynetskiy and fellow clinical faculty at the School of Pharmacy, clinicians at Temple University Hospital and Jeannes Hospital. The researchers are studying why people process the same drug differently. In this case, they're trying to find the correlation between genotypes, or a person's inner code of DNA, and the correct dosage of Warfarin. By collecting saliva samples and extracting DNA from 77 participants already on the drug, the researchers can look for variances, genetic clues, which make people metabolise the same drug in very different ways.
"Our findings have confirmed there is a genetic variance of certain genotypes that correlate to how these participants respond to this drug," says co-investigator Nima Patel, associate professor in the School of Pharmacy. "So, if you have this genotype, we can conclude what your risks may be, based on your DNA."
That would allow doctors to prescribe the correct dosage of Warfarin and decrease the risk of adverse drug reactions: too low a dose can increase the risk of dangerous blood clots, while too large can cause life-threatening bleeding. What may be equally noteworthy about Krynetskiy's and Patel's research is that more than half the participants are either African American or Hispanic, two groups underrepresented in clinical trials. So finding their therapeutic window, the place where they will safely get the maximum benefit of a drug, is particularly important in this personalised medicine quest.
