Matthews Lecture 2_final 140115

Dna analyze by pcr or other means 36 chromatin

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Unformatted text preview: breast - uterus + bone + breast - uterus - bone + breast - uterus ? bone ? Brzozowski et al. Nature 1997 Pike et al. EMBO J 199933 Ligands influence coregulator complex composi/on Chroma/n immunoprecipita/on assay (ChIP) •  resulted from the pioneering efforts of several researchers in the mid 80s and early 90s who used protein cross- linking in chroma/n analysis •  valuable technique that allows researchers to establish when and which proteins bind to in vivo DNA sequences •  provides a snapshot of the na/ve chroma/n structure and the factors bound to genes in different func/onal states •  allows for the study of the effects of histone modifica/ons on transcrip/on in vivo 35 Chroma/n immunoprecipita/on assay (ChIP) •  has been used in yeast, drosophila, rodents and mammalian cell lines •  ChIP assays involve the following: •  trea/ng cells or /ssues briefly with formaldehyde •  fragmen/ng the DNA/protein complexes •  immunoprecipita/on •  reversing cross- links and isola/ng DNA •  analyze by PCR or other means 36 Chromatin immunoprecipitation An/body bonds specific transcrip/on factor Immunoprecipitate with an/body Collect chroma/n- an/body complex Treat cells with formaldehyde Sonicate to produce fragments of chroma/n Reverse cross links Purify DNA Fragments of chroma/n with transcrip/on factors cross- linked to DNA DNA fragment containing specific transcrip/on factor binding site by PCR or qPCR 37 Advantages/Disadvantages of the ChIP Assay •  requires a specific an/body; d...
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