A comparative analysis of shotgun-cloning and tagged-random amplification-cloning of chromatin immunoprecipitation-isolated genome fragments.
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.
White
Robert B
RB
School of Exercise, Biomedical and Health Science, Edith Cowan University, Joondalup Drive, WA, Australia.
Ziman
Melanie R
MR
D000818:Animals; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry; D004622:Embryo, Mammalian / Q000378:metabolism; D016678:Genome; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D014157:Transcription Factors; D004247:DNA