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Portland, OR, United States

Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 0.00 | Year: 2005

DESCRIPTION (provided by applicant): 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.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 959.36K | Year: 2005

DESCRIPTION (provided by applicant): 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.


Trademark
Portland Bioscience Inc. | Date: 2007-10-04

Computer hardware for research and scientific use concerning biotechnics, gene technology and DNA-analysis; Computer software for scientific analysis of genetic sequences, genetic information and biological materials for use in research in the fields of genomics, biology, chemistry and medicine; DNA chips; Biosensors for research and scientific use concerning biotechnics, gene technology and DNA-analysis.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 170.50K | Year: 2007

DESCRIPTION (provided by applicant): This is a SBIR Fast-Track application to address two currently unmet needs. These are for a quantitative theoretical understanding of DNA multiplex hybridization reactions; and a general design methodology for developin g rapid, reliable, multiplex genotyping assays. First, in a broader sense there is presently a void in theoretical understanding of DNA multiplex hybridization. For example, current analytical understanding of the thermodynamics and kinetics of the multipl ex microarray hybridization process is minimal. Plans in Phase I include development and implementation of our theoretical multiplex analytical methods, and experimentally demonstrate feasibility of using temperature dependent kinetics to discriminate a pe rfect match duplex from one containing a single base pair mismatch, i.e. SNP, on a microarray. If feasibility can be established, the first part of Phase II is focused on quantitative evaluation of essential experimental parameters, and further parameteriz ation of the theoretical model. This will establish a solid and more rigorous analytical basis for understanding DNA multiplex hybridization reactions. This theoretical foundation will immediately enable more insightful design, more accurate analysis, and greatly expanded and enhanced predictive capabilities for novel and improved design of microarray based assays. This analytical foundation will also support development and applications of novel diagnostic microarray formats that incorporate and utilize ti me and temperature as key assay parameters. In the second part of Phase II, to demonstrate capability and immediate utility of our analytical process, we will design, build, test and validate a rapid genotyping panel test for 11 single nucleotide polymorph isms in eight genes that have been associated with Adult Macular Degeneration, a leading cause of age related vision loss. The developed process can be generally applied to the design of multiplex genotyping assays for the detection of virtually any SNP pa nel for any standard microarray platform. Principal Investigator: Benight, Albert S. Project Narrative The goal of this SBIR Fast-Track project is to develop new and powerful analytical tools that will be parameterized and implemented to provide capabiliti es of quantitative modeling of the temperature dependence of DNA multiplex hybridization kinetics on a microarrray. The developed tools will be readily applicable to address prescient needs in custom diagnostic assay design. A demonstration of the develope d technology will be a rapid and cheap genotyping assay for detection of Single Nucleotide Polymorphisms (SNPs) that may be associated with Adult Macular Degeneration, a genetic disease responsible for nearly 50% of age related vision loss in developed cou ntries.


Williams L.,University of Utah | Blair S.,University of Utah | Chagovetz A.,University of Utah | Fish D.J.,Portland Bioscience Inc. | Benight A.S.,Portland Bioscience Inc.
Analytical Biochemistry | Year: 2011

Under equilibrium conditions, there are two regimes of target capture on a surface - target limited and probe limited. In the probe limited regime, the melting curve from multiplex target dissociation from the surface exhibits a single transition due to a reverse displacement mechanism of the low affinity species. The melting curve cannot be used in analytical methods to resolve heteroduplexes; only with the simplex system can proper thermodynamics be obtained. © 2010 Elsevier Inc. All rights reserved. Source

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