Home    ||    About DOC    ||    Hardware    ||    Software    ||    Applications & Demonstrations    ||    Data Access Portal   ||  Methods    ||    References & Papers

About DOC

Background

Parallel expression, mutation and genotyping analysis for many genes within a living system has become possible via a number of technologies ­ automated DNA sequencing, mass spectroscopic methods and microarrays.  These technologies make it possible for us to identify new opportunities for understanding that lead to new knowledge, medical diagnostics, and therapeutic drugs.    They are of greatest utility for the genomes of organisms for which the most sequence data are available, especially humans and commonly used model organisms for human disease, like mice and rats.    The availability of the complete genome sequence enables us to annotate the genes and other important components of their genomes, such as regulatory loci.  One can then analyze their gene components simultaneously, quickly, efficiently and cost effectively.  One of the most promising technologies in wide use are DNA microarrays which come in many forms, from nanoliter spots that are robotically deposited to microchips made using techniques borrowed from the semiconductor industry or ink-jet printer technology.  Two industry leaders have emerged, Affymetrix (Santa Clara, CA) and Agilent Technologies (San Jose, CA).  Each has their own approach, but both focus, at least for the time being, on measuring the expression level of massive collections of genes on their chips.  Gene expression in a given cell type (for example, normal and cancerous cells) under a given environmental condition can be a good indication to a gene’s potential biomedical role, and therefore of valuable for finding new drug leads, thus the scientific and commercial popularity of expression microarrays.

Custom Microarray manufacturing technologies

Despite our ability to make many thousands of measurements simultaneously, the enormous size of the genome necessitates careful target selection to tune experiments to address specific questions. A need has emerged to have rapidly customizable microarrays.  There are two dominant methods to accomplish custom microarray synthesis: Affymetrix-like microarrays manufactured using light projection rather than semiconductor-like masks used by Affymetrix to mass manufacture their GeneChip^TM arrays, or the ink-jet printing method employed by Agilent.  The manufacture of custom Affymetrix-like microarrays is done on a Digital Optical Chemistry (DOC) machine developed at the University of Texas Southwestern Medical Center, and this method offers higher feature density than possible with ink-jet printed arrays.  The initial DOC prototype has been described in several publications and has led to two second-generation machines.  Each machine reliably produces three arrays daily, has been deployed against a number of biomedical questions, is being used in new ways, and has led to a number of spin off technologies.  Much of the software required for this project has been demonstrated to be of independent value and these packages have been made available through the www.

            The DOC devices produces chips having up to 196,000 genetic features.  The heart of these machines is the Texas Instruments Digital Light Processor (DLP^TM), the key component in most of today’s computer projectors.  The DLP^TM modulates the light intensity it projects by independently canting its nearly 800,000 micromirrors (17 microns on a side) under computer control.  Unlike liquid crystal light modulators, the DLP^TM can reflect ultraviolet light at 365 nm, a wavelength where available high intensity mercury UV light sources emit and where the photo-reactive groups on the DNA chemical precursors have a high cross section.  Merging the DLP^TM projection system with an enhanced version of the fluidics system built for a high throughput DNA synthesizer, called the MerMade^TM (developed by H. Garner, donated to the public domain and made available from BioAutomation, Plano, TX) enables one to manufacture a custom high-quality DNA microarray in less than 8 hours.   This device needs only a chemically treated microscope slide, the light activated chemicals, and a data file containing the 196,000 DNA sequences to make a new chip.  Each array includes control features that serve as diagnostic indicators of chip quality with a labeled control oligonucleotide added to every hybridization sample. The control features test reproducibility of hybridization signal and discrimination in binding against single-nucleotide mismatches. Signal from perfect match hybridization to the control oligonucleotide varied by less than 25% over all chips and all queried sequence of the control oligonucleotide could be read correctly on every chip.

Left: Picture of 2^nd generation DOC Instrument, one of 2.  Right: A close up picture of the optics, fluidics and reagents.  The second-generation Digital Optical Chemistry synthesizer, was constructed using lessons learned in the prototype device.  The DOC 2 device incorporates improved fluidics and optics, and additional operations software to enhance the ease with which different array designs can be made for different functionality.

With these new machines in place, arrays of arbitrary design are fabricated quickly, so the bottleneck shifts to the chip design and analysis. To address the design of these chips, a substantial amount of bioinformatics software was written for each of the applications for the device.   While the technology can produce the popular gene expression chips, those are available commercially from Affymetrix, are inexpensive, have high gene coverage and high quality.  DOC can manufacture expression arrays for genes and genomes not available on Affymetrix GeneChips^TM.  So, this technology is best suited for other applications.  For cancer applications, the re-sequencing of tumor suppressor genes, oncogenes or their regulatory regions is ideal for the DOC technology.  As more and more cancer-relevant genes are identified, these custom chips can readily be expanded or changed to incorporate them.  Recently, the methylation of specific DNA bases in the regulatory regions of genes has shown to play a major role in the genetics/epigenetics of cancer, methylation-specific DOC arrays and methods were developed for that analysis.  Further, many cancers show deletions of genetic material of various sizes, including many genes, and this technology has manufactured genome scanning arrays (CGH) to measure these phenomena.  Many types of measurements that can be made with a flexible system, and these methods are of value for other biomedical studies, such as epigenetic disease research, anti biological warfare measures and agricultural applications.