SBIR-STTR Award

Hybridization of nucleic acid mixtures to surfaces containing attached complementary nucleic acid moieties (multiplex hybridization) is the principal step in nucleic acid based microarray technologies. Multiplex microarray technologies are increasingly becoming the premiere tool for measuring genome wide gene expression and many diagnostic and prognostic applications in: basic research, drug discovery/development, and assessment of disease susceptibility, therapy strategies, and monitoring. Microarray technology can increase productivity of current low throughput biological assays by hundreds to thousands of times. Such gains hold promise for reducing costs, and broadening availability of such testing to the public. Key benefits arising from this are the possibility for pre-disease treatment protocols. Reliability factors such as high sensitivity and accuracy are major keys to enabling array technology use in all of these scenarios. The proposed research program will test our technology's ability to eliminate the effects of phenomena occurring at the intra and inter molecular level in oligonucleotide DNA microarray hybridizations that: a) reduce sensitivity, precision, accuracy, reproducibility, comparability, and b) increase array density needed, background "noise", assay cost, reliance on "image analysis software interpretations" in any DNA multiplex hybridization based microarray based assay. PROPOSED COMMERCIAL APPLICATIONS: The research will be the first step in developing Chemical standards for designing multiplex DNA Arrays enabling less costly, highest fidelity assays and reliable cross-platform data comparisons. This set of features will be useful in designing unique assays for each "facet" of the health care sector as mentioned above. We believe this broad market can be served because a strategy of licensing of assay chemistries would be pursed. We would stay focused on assay chemistry technology while licensees handle the hardware and software engineering, marketing and system service.

Multiplex hybridization of a number of nucleic acid single strands resulting in the formation of many sequence specific, individual duplex complexes, is the central process in a number of nucleic acid based diagnostic assays. Such assays are currently employed to analyze gene expression, detect genetic variations and mutations, assess viral loads in response to therapeutic agents and detect infectious pathogenic agents. Ideally, each strand present in a multiplex reaction is meant to form a duplex with only its perfectly matched and complementary single strand. In reality however, depending on the particular sequences present, many of the resident single strands can also anneal with strands other than their perfectly matched complement strand, resulting in cross-hybridization, x-hyb. X-hyb is a major nemesis of multiplex hybridization reactions causing false positive signals, lowered accuracy and sensitivity. Although x-hyb is readily acknowledged as a potential problem by all practicitioners of multiplex hybridization assay, the molecular interactions responsible for x-hyb are not understood. Other than redesigning the probes and primers and trying them again (very much a trial and error, empirical approach) there is little recourse should a given probe and/or primer set fail to function as designed [due to x-hyb]. The goal of this Phase II project is to quantitatively define sequence dependent features of x-hyb and develop a robust analytical framework for diagnosing and predicting x-hyb on the level of individual sequences. The first specific aim involves evaluation of thermodynamic parameters for tandem mismatches as a function of sequence, in solution and on microarrays, then incorporating this information in a software toolset for x-hyb analysis. The second specific aim is to apply the capabilities gained to develop RT-PCR and microarray based assays for detection of an alternatly spliced variant of the Creld1 gene, the (exon 9b) isoform, whose presence has been has been linked to various cancers.

Thesaurus Terms:
Microarray Technology, Nucleic Acid Hybridization, Nucleic Acid Sequence, Technology /Technique Development Computer Program /Software, Computer System Design /Evaluation, Gene Expression, Molecular Biology Information System, Thermodynamics Human Tissue