SBIR-STTR Award

Changes in methylation state and DNA copy number within the genome play a key role in cancer development and progression. Localization and quantification of these changes is important to the discovery of new tumor suppressor genes and oncogenes, and offers great potential in classifying cancer for clinical disease management. As such, the overall objective of this project is to develop a high-throughput array-based commercial technology to globally scan the genome for changes in DNA copy number (goal to detect two-fold differences) and methylation state. This will be accomplished via a reduced complexity representation approach using comparative genomic hybridization (CGH) and restriction landmark genome scanning (RLGS) technology on a BeadArrayTM platform. There are several immediate benefits offered by this representation approach. First of all, amplification of the representations will enhance signal to noise on the array. Secondly, improvements in generating representations will provide a more reproducible and robust process, allowing accurate detection of DNA copy number and methylation changes especially from archival samples exhibiting DNA degradation. Finally by converting RLGS from a 2-D gel-based approach (approximately 1000-2000 loci) to a BeadArrayTM-based analysis (approximately 1000-50,000 loci), a much higher locus resolution and sample throughput will be realized. This should enable the large-scale analysis of hundreds to thousands of tumor samples and lead to improved understanding of tumorogenesis. Phase II will apply this technology to the analysis of tumor samples and cell lines.

Thesaurus Terms:
DNA methylation, DNA replication, functional /structural genomics, genome, high throughput technology, representational difference analysis, technology /technique development experimental design, genetic model, model design /development, restriction endonuclease, subtraction hybridization human genetic material tag, microarray technology, polymerase chain reaction

Anomalies of the genome, such as congenital chromosomal imbalances, can be present from birth or can arise during cell division and lead to formation of cancer. Progression of cancer can be followed by monitoring changes in the genome. For instance, allelic deletions of tumor suppressor genes and amplifications of oncogenes are well known events in carcinogenesis. The primary goal of this project is to develop a high resolution array-based approach to detecting and monitoring changes in tumor samples. A secondary goal is to detect microdeletions and amplifications in congenital chromosomal disorders. The proposed work will focus on developing DNA copy and loss of heterozygosity (LOH) measurements using our novel whole genome genotyping (WGG) platform. The WGG technology was developed, in part, through phase I funding. Under phase I, we developed a whole genome amplification protocol and an arraybased primer extension assay that enabled direct readout of the genome - both copy number and genotypes. This development obviates the need for the original proposed complexity reduction step (reduced representation). The number of genotypes that can be read out from a single sample is limited only by the number of probes on the array. The proposed work will use a WGG array that is currently under development to perform genotyping and copy number analysis at the same time, at an average resolution of approximately 30 kb across the genome. This revolutionary assay system will allow regions of genomic alteration to be precisely defined accurately and robustly, and has the potential to be used directly in clinical applications. Once fully developed, the assay will be employed to characterize biological tumor/normal sample pairs as well as congenital chromosomal imbalances