Single-Base Resolution Method of 5-hmC in Biology

Award Information
Agency:
Department of Health and Human Services
Branch
n/a
Amount:
$212,188.00
Award Year:
2012
Program:
SBIR
Phase:
Phase I
Contract:
1R43HG006634-01
Agency Tracking Number:
R43HG006634
Solicitation Year:
2012
Solicitation Topic Code:
NHGRI
Solicitation Number:
PA11-096
Small Business Information
CINLANIAN BIOTECHNOLOGY, INC.
4095 CLAIRMONT ROAD, ATLANTA, GA, 30341-
Hubzone Owned:
N
Socially and Economically Disadvantaged:
N
Woman Owned:
N
Duns:
965940815
Principal Investigator:
SHUANG CHANG
(301) 318-3650
rnaie2005@gmail.com
Business Contact:
WEI XHANG
(301) 318-3650
wei_zhang@cinlanian.com
Research Institution:
Stub




Abstract
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.

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