I ran across Health Reseach Inc.'s Technology Transfer website and thought that maybe somone would be interested in some of these opportunities. For the full list go to:

http://www.hrinet.org/



Technology Opportunity

Invention Available for Licensing and Commercialization




Tag-Adapted Bisulfite Genomic Sequencing for Fine-Mapping of DNA Methylation




Description



A new method for assaying methylation of cytosines at CpG dinucleotides has been developed. The PCR-based method allows for continuous, fine mapping of DNA methylation, without the need for prior knowledge of the methylation status of a CpG site, or requirement for DNA cloning. The methods reliability and simplicity mark this as an important technical advance for biological studies.



Epigenetic regulation of DNA structure, and consequent functional impacts on promoter-regulated expression, silencing and other functions, has been a major focus of biological research over the last 15 years. Studies of gene regulation, development, and carcinogenesis have been particularly impacted by the recognition of the import of DNA cytosine methylation, when in the 5-CpG-3 configuration.



A technical limitation to these studies has been the inability to obtain precise, reliable DNA sequence, without cloning PCR fragments into bacteria. The standard approach is to use bisulfite chemical conversion of unmethylated cytosine residues, specifically, to uracil, and subsequent substitution with thymidine on subsequent cycles of PCR. Methylated cytosines are resistant to bisulfite conversion, and remain cytosines in the PCR. This approach serves as the basis for both bisulfite genomic sequencing (BGS, Frommer et. al.), and methylation-specific PCR (MSP, Herman et. al.). For BGS, direct cycle sequencing of the PCR product is performed if possible. Unfortunately, it is often difficult to sequence the resulting PCR product without cloning the PCR product. For MSP, specific primers can be designed within considerable constraints, with prior knowledge of a CpG sites likely methylation status, as only one or two CpG sites can be probed in any given MSP reaction. Sensitivity and specificity of the assay at any given residue is therefore highly dependent on primer design, and often suboptimal. Additionally, the MSP approach yields methylation maps that are therefore discontinuous.



Scientists have now generated a method by which unmethylated cytosines can be distinguished from methylated cytosines reliably, at a base-by-base fine-mapping resolution, employing PCR and direct cycle sequencing only, without method-specific constraints on primer design parameters, and obviating the need for prior knowledge of likely methylation status as well as DNA cloning.



Potential Areas of Application: Any biological, medical or epidemiologic inquiry into DNA methylation, such as:

Gene regulation research

Developmental research

Inquiries of prokaryotic genomes

Epigenetic studies

In-vivo expression phenotyping

Carcinogenesis

Cancer detection

Defining at-risk populations


This diagnostic method monitors changes in viral tropism associated with HIV disease progression, and can be used as a diagnostic tool to monitor the effectiveness of antiviral therapy and disease progression in HIV positive individuals.



A change in viral tropism occurs in many HIV positive individuals over time, and this change is indicated by a shift in coreceptor usage that has been shown to correlate with increased disease pathogenesis. Therefore, application of the diagnostic method to monitor shifts in coreceptor usage is useful for predicting disease progression.



Understanding coreceptor usage during potent antiretroviral therapy is relevant to HIV-1 dynamics and the maintenance of viral suppression and clinical response. The method utilizes a mathematical model to measure the proportion of virus in a specimen that uses each coreceptor. Coreceptor utilization studies may have a role in virologic monitoring of patients on antiviral therapy, particularly as drugs are developed which target R5 or X4 strains of HIV-1. The diagnostic method and the mathematical model provide a laboratory technique to monitor the effectiveness of antiviral therapy.

Main Advantages:

1. Allows for fine-mapping, at single base resolution, of CpG-DNA methylation.
2. Highly reproducible results.
3. User friendly, not labor intensive.
4. Sensitive, PCR-based approach.
5. No additional PCR-primer constraints.
6. No DNA cloning required.
7. No prior knowledge of likely methylation status at a CpG residue is required.
8. Compatible with most direct cycle sequencing chemistries and platforms.



Potential Market Applications:

* Genetic research.
* Gene expression or developmental research.
* Epigenetic research.
* Studies of gene-environment interaction.
* In-vivo expression phenotyping.
* Carcinogenesis.
* Cancer detection.
* Defining at-risk populations.





State of Development:

1. Research level validation of method in benign and malignant human cells.
2. High volume validation in 12 DNA fragments in duplicate, across 2 genes and >40 human peripheral blood specimens.
3. Validation in exfoliated human cytologic specimens.










Licensing Potential

HRI seeks commercial partners to develop the technology for research and clinical use. Available for licensing.



Contact:



Bob Gallo

Associate Director Technology Transfer

Health Research, Inc.

One University Place

Rensselaer, NY 12144-3456

(518) 431-1208 Fax (518) 431-1234
rlg04@health.state.ny.us






References:

Herman JG, Graff JR, Myohanen S, et.al. Methylations specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl Acad Sci, USA, 93: 9821-26, 1996.



Frommer M, McDonald LF, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci, USA, 89:1827-1831, 1992.