A Comparative Analysis of Shotgun-Cloning and Tagged-Random Amplification-Cloning of Chromatin Immunoprecipitation-Isolated Genome Fragments
Document Type
Journal Article
Keywords
Target gene identification, Pax7, T-PCR, ChIP
Publisher
Academic Press Inc Elsevier Science
Faculty
Faculty of Computing, Health and Science
School
School of Exercise, Biomedical and Health Science
RAS ID
5248
Abstract
The cloning of transcription factor antibody-immunoprecipitated genomic fragments from chromatin immunoprecipitation (ChIP) experiments is a technically challenging procedure, especially when the input genomic DNA is isolated from whole tissues (in vivo) rather than cultured cells. Here we adapt a technique known as Tagged-Random PCR (T-PCR) to amplify ChIP-immunoprecipitated DNA from mouse embryonic tissue prior to cloning. Importantly, we then compare this technique with tandem shotgun-cloning experiments in terms of its capacity to identify target genes. We find that T-PCR dramatically increases the efficiency of cloning ChIP fragments without distortion of the relative location of cloned fragments to putative target genes. Thus, T-PCR is a simple procedure which greatly enhances the efficiency of cloning tissue-derived ChIP fragments.
Comments
White, R. B., & Ziman, M. R. (2006). A comparative analysis of shotgun-cloning and tagged-random amplification-cloning of chromatin immunoprecipitation-isolated genome fragments. Biochemical and Biophysical Research Communications, 346(2), 479-483. Available here