The requirement to redesign and re-optimise the entire set of PCR assays if only one target is changed imposes a severe penalty on experiment flexibility, particularly as the number of multiplexed assays increases. PCR in a miniaturised serial or parallel fluidics systems is the final strategic option. Continuous flow monolithic lab-on-a-chip devices allow rapid sequential PCR amplification and analysis of micro- or nanolitre samples in a micromachined channel passed through multiple, fixed temperature zones followed by detection of the amplified target sequences.
Sample throughput is limited by sampling spacing in the channel to prevent cross-contamination and problems endemic to serial microfluidic systems such as microchannel clogging and inefficient interfaces to microplates, the standard fluidics carrier in pharmaceutical drug discovery. Although a number of experimental systems have been reported1, the technology has matured to the point where there are several commercial high throughput products for quantitative real-time PCR.
Book Microarray Innovations: Technology And Experimentation (Drug Discovery Series)
Fluidigm www. A dynamic array is an integrated fluidic circuit IFC comprised of an array of miniature pneumatically-actuated valves that form a high density array of nanolitre reaction containers when actuated.
Available in either 48 x 48 or 96 x 96 array formats, sample and qPCR reagents are dispensed into access ports compatible with fluidic transfer from a SBS-standard microplate. Actuation of the valves allows 48 samples to be tested for 48 gene transcripts qPCR tests or 96 samples against 96 transcripts qPCR tests in 6. Roche Applied Sciences www.
Samples and reagents in standard microplates are robotically combined and thermally cycled in 0. Reagent savings and assay-sample customisation are important strengths of these systems.
BioTrove Inc www. The gene assay panels are organised to allow quantitative, system-level investigations of expression profiles across different diseases eg cancer, cardiovascular, diabetes , mechanisms of biological action eg apoptosis, signal transduction, toxicity or gene families kinome for human, rat and mouse specimens. The spatial independence of each qPCR assay allows a high degree of assay multiplexing without compromising data quality or assay performance.
Furthermore, assay independence means optimised semi-custom or custom assay panels are generated by replacing specific assays or adding to the panel without re-design of the existing assays. Fluidic management via passive capillary action makes the system reliable, robust and greatly simplifies workflow integration with SBSstandard microplates.
Automating Drug Discovery’s Next Breakthrough
More predictive, biologically-relevant high throughput screening techniques are necessary to improve early stage compound screening. Rejecting compounds early in development based on actionable, high quality safety and efficacy data from assays relevant to the target disease and human physiology will bring substantial benefits by avoiding the much higher expense of failure down the line in human clinical trials. Many high-throughput screens involve solutionphase biochemical assays in which compounds are tested for biological activity against a selected enzymatic target.
An example of a biochemical assay is a kinase assay in which the ability of a test compound to attenuate the incorporation of one or more phosphate groups into a protein or peptide substrate is monitored. There are several methods by which the phosphopeptide product can be quantified, including the use of radiolabelled phosphate, ELISA-based methods, or a derivatised substrate incorporating a fluorescent label 5.
In many cases enzymatic targets are not amenable to conventional assay methods due to the characteristics of the substrates and products involved. In other cases, an economically viable HTS assay cannot be developed. In either event, potentially valuable targets are often not pursued. As a consequence compromises are made in assay design that lead to suboptimal screens in which biologically active compounds are not detected.
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Envisioning the Future: Medicine in the Year 2050
All stages All stages Submission open Submission closed Ebook available. Eschewing the temptation to commercialize its success, Brown's lab posted instructions for building a homemade spot-printing robot on its Web site. Before long, the do-it-yourself microarray movement had taken hold in many academic labs, where researchers wanted custom slides without the high cost of commercial arrays, and were willing to tinker to get results. These days commercial chips have become inexpensive enough, and their quality, reliability, and variety good enough, that labs just breaking into microarrays might be better served by commercial rather than by homegrown chips.
Still, spot printing remains a relatively cheap and flexible option for many labs and core facilities. Surprisingly, neither Affymetrix's in situ manufactured chips nor its competitors' spot-printed arrays has been declared a clear winner in terms of accuracy and reproducibility of results. Several studies, most recently a series in Nature Methods , 5 have found that both approaches are prone to error and misinterpretation, and both can deliver good results in experienced hands.
Designed to screen patients for genetic variations in drug metabolism, the array is the first approved by the Food and Drug Administration for use as a diagnostic.
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Other such products are in the pipeline, as are gene-expression based arrays, which have been used in research labs to stratify cancers and predict clinical outcome. But a lack of standardized protocols for experimentation and analysis is stymying progress. Where most of the noise comes in is, how do you collect your sample, how do you prepare your sample, and how do you do your experiment? Several efforts are underway to fill the standards gap.
Another project, the External RNA Controls Consortium, is developing a set of "spike-in" RNAs that can be added directly to experimental RNA samples as external controls to monitor the performance of microarray processes. Though such controls and benchmarks may ultimately be required of labs that wish to run gene-expression analyses for diagnostic purposes, or for companies submitting array data to the FDA, they remain, for the moment, research projects, says Leming Shi, MAQC coordinator. Meanwhile, other companies, like Agilent Technologies and Oxford Gene Technologies, have refined and capitalized on inkjet printing methods, bolstered by interest from giants like Hewlett-Packard and Merck.
Still others, such as CombiMatrix, have harnessed developments in microfluidics to find new ways of generating microarrays. Nevertheless Affymetrix retains the lion's share of the microarray market — and it fights hard to keep it. Beginning with a pitched battle over the Southern patents, the company has been embroiled in a string of intellectual property lawsuits and disputes over licensing agreements. One source of perpetual consternation to its competitors is the fact that Affymetrix claims its patents cover not only the photolithographic technique, but also the high density of its arrays as well.
To a large extent, Affymetrix's position on that front is secured by both patents and physics, as an array's density is a function of the technology used to create it. Inkjet and spotting methods are constrained to a maximum of about 50, features on a slide by the minimum size of a droplet, whereas features constructed with semiconductor technology are constrained only by the size of a manipulable beam of light. The most recent generation of Affymetrix chips has six million features, each five microns across, and the company has prototype chips with one-micron features.
With such vast numbers of features per chip, researchers are free to consider studies never before possible. Scientists armed with "genomes-on-a-chip" can now easily scan the expression of hundreds of thousands of genes for the few that might be involved in a form of cancer or a genetic disorder.