DNA microarray technology has become one of the most widely used tools for functional genomics and is playing an ever increasing role in the study of viral infections and host-pathogen interactions. With Dr. Hua Zhu of the New Jersey Medical School we have recently developed an oligonucleotide microarray representing all the predicted open reading frames of the human cytomegalovirus (HCMV) and an established protocol for simultaneously measuring the expression of all HCMV genes. To evaluate the performance of the HCMV array, human foreskin fibroblasts were either mock infected or infected with the HCMV AD169 or Toledo strains. Hybridizations were performed to determine the level of detection of HCMV transcripts from both the AD169 and Toledo strains and to assess reproducibility within and between slides. Overall, approximately 95% of the predicted HCMV genes produced detectable levels of mRNA, with median signal to noise and signal to background ratios of 41 and 14 respectively. Scatter plots of samples within an array and between two arrays resulted in average linear regressions above 0.95 and 0.9 respectively, indicating that data from the arrays are highly reproducible. In addition, transcripts from genes found in the Toledo strain but not in AD169 were specifically detected.
For details on the HCMV microarray, read our paper Using DNA microarray to study human cytomegalovirus gene expression.
The application of microarray technology for diagnostic mutation detection testing has the potential to change molecular genetics and personalized medicine. The simultaneous analysis of multiple mutations is expected to not only decrease the turnover time in the lab but also decrease the cost per genotype. Genotyping assays have been developed by several companies however they generally require that end users utilize the reagents, arrays and equipment from their company. Small specialized panels which focus on minority or ethnic groups are often not being developed due to their lack of profitability. This results in the need to use multiple platforms to perform several different tests. Together with the laboratory of our collaborator Dr. James Dermody at the New Jersey Medical School, we have recently developed a spotted array-based mutation testing system which is compatible with most of the commercially available microarray scanners and reagents. This array consists of 32 alleles for 16 of the most common genetic diseases affecting the Ashkenazi Jewish population. The basis of this assay is the hybridization of a fluorescently labeled multiplex PCR reaction to a microarray spotted with 25-mer oligonucleotides representing the wild-type and mutant allele for each mutation. Independent samples are scored using a linear discriminant analysis (trained on a training set) into homozygous wild type, heterozygous or homozygous mutant for each mutation.
For more information on this project see our poster A spotted array-based open mutation testing system presented at the 8th International Meeting of the Microarray Gene Expression Data Society.
Recent advances in protein microarray technology have shown its great potential as a powerful and versatile tool for large-scale studies of protein function. Protein arrays have demonstrated their utility for analyzing enzymatic activities, antibody-antigen, protein-protein and protein-DNA interactions. However, there are many significant hurdles that must be overcome before protein arrays will gain widespread use. Most important among these are a reduction in the cost of producing the arrays and the development of surfaces that maintain protein structure and function. Together with our collaborator Dr. Matthew Libera at the Stevens Institute of Technology we are developing an innovative new array surface based on nanohydrogels that can scale microarray technology into the nano regime. Using focused electron beams, nanohydrogels of approximately 200 nm in diameter have been generated from amine-terminated poly(ethylene glycol). The nanohydrogels can be patterned on glass or silicon at submicron spacing, with approximately 7500 nanohydrogels in a 100 micron diameter spot. Using a nucleic acid binding assay we have demonstrated the attachment and retention of function of a nucleic acid binding protein. The nanoarray exhibits superior performance to equivalent microarrays produced using conventional microarray printing technology.
To learn more about this project see our poster Ultra-high Density Hydrogel Nanoarrays presented at Chips to Hits 2005.
