Detection of Genetic Lesions in Breast Cancer
Abstract
The purpose of this proposal is to develop a "next generation" representational difference analysis protocol that uses transcribing nuclear DNA. This protocol should detect all transcribe genes aberrantly transcribed during the progression of breast cancer. Genes, for example, that produce strictly nuclear RNA should be isolated by this protocol. Our first specific aim was to isolate transcriptionally active nuclear DNA from human breast cancer cells. Features that distinguish transcribing chromatin from bulk chromatin are unfolded nucleosomes with thiol-reactive histone H3 and highly acetylated H3. A new method using Sulfolink column chromatography has been developed to isolate transcribing chromatin with thiol-reactive H3 from T47D5 breast cancer cells. Further, we are in the process of developing an alternate procedure for the isolation of transcriptionally active chromatin by a chromatin immunoprecipitation approach. Here we use anti-acetylated H3 antibodies to immunoprecipitate highly acetylated H3 that is cross-linked to DNA fragments. We show that both methods will isolate transcriptionally active chromatin.
Document Details
- Document Type
- Technical Report
- Publication Date
- Sep 01, 1998
- Accession Number
- ADA365469
Entities
People
- James R. Davie
Organizations
- University of Manitoba