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 chemically modified 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 approach can 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 only have great values as commercial research reagents, but also promise to efficiently discover and detect 5-hmC as biomarkers in various human diseases. !
5-Hydroxymethylcytosine (5-hmC) is a newly discovered base modification surprisingly 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.