Single-Base Resolution Method of 5-hmC in Biology

Award Information
Agency: Department of Health and Human Services
Branch: N/A
Contract: 1R43HG006634-01
Agency Tracking Number: R43HG006634
Amount: $212,188.00
Phase: Phase I
Program: SBIR
Awards Year: 2012
Solicitation Year: 2012
Solicitation Topic Code: NHGRI
Solicitation Number: PA11-096
Small Business Information
DUNS: 965940815
HUBZone Owned: N
Woman Owned: N
Socially and Economically Disadvantaged: N
Principal Investigator
 (301) 318-3650
Business Contact
Phone: (301) 318-3650
Research Institution
DESCRIPTION (provided by applicant): 5-Hydroxymethylcytosine (5-hmC) is a newly identified base modification in mammalian genomic DNA. Because current sequencing methods cannot differentiate 5-meC from 5-hmC, the immediate challenge is to develop robust methods to ascertain the positions of 5-hmC within the mammalian genome, a problem best addressed by adapting a new chemical labeling technology that we have developed. We show that the hydroxymethyl group of 5-hmC can be selectively labeled with chemicallymodified glucoses using 2-glucosyltransferase (2GT). This glycosylation offers a strategy of installing functional groups such as biotin onto 5-hmC. In this way, we can affinity capture DNA fragments containing the modified 5-hmC and develop sequencing methods to determine the precise locations of 5-hmC. Building on our early successes we propose to develop single base-resolution detection and sequencing methods to reveal the distribution of 5-hmC in mammalian genomes. We propose two different approaches, both utilizing the selective chemical labeling strategy we have developed. In one approach, we will label 5-hmC with bulky groups to hinder ligation and linear PCR reactions, thus achieving single base-resolution detection of 5-hmC. The linear PCR approachcan be adapted into the next generation Solexa sequencing to perform high throughput determination of 5-hmC in mammalian genomes. In an alternative approach, we wil develop an exonuclease digestion blockage method that detects modified 5-hmC at 3' end of undigested DNA fragments using the next generation Solexa sequencing; we have already shown that the chemically modified 5-hmC blocks exonuclease III digestion at 3' end of the modified 5-hmC. The new technologies proposed in this SBIR application not onlyhave great values as commercial research reagents, but also promise to efficiently discover and detect 5-hmC as biomarkers in various human diseases. PUBLIC HEALTH RELEVANCE: 5-Hydroxymethylcytosine (5-hmC) is a newly discovered base modificationsurprisingly abundant in the genomic DNAs of certain mammalian tissues and cells. The proposed work will develop efficient chemical labeling methods to perform single base-resolution detection and sequencing of 5-hmC in genomic DNAs. The success of the proposed work will enable the researchers to reveal the fundamental role(s) of 5-hmC in biology and develop potential disease markers.

* Information listed above is at the time of submission. *

Agency Micro-sites

SBA logo
Department of Agriculture logo
Department of Commerce logo
Department of Defense logo
Department of Education logo
Department of Energy logo
Department of Health and Human Services logo
Department of Homeland Security logo
Department of Transportation logo
Environmental Protection Agency logo
National Aeronautics and Space Administration logo
National Science Foundation logo
US Flag An Official Website of the United States Government