ChIP-on-Chip

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A ChIP-on-Chip or ChIP-Chip ( Chromatin-Immunoprecipitation Chip ) is a biochemical method for determining protein-DNA interactions . The ChIP-on-Chip is a combination of chromatin immunoprecipitation and hybridization with a DNA microarray (synonym DNA chip ). DNA-protein interactions occur in binding sequences for transcription factors , promoters , enhancers , repressors , silencers , isolators and in binding sequences for DNA replication .

properties

The purified recombinant protein is either mixed with the purified DNA ( in vitro ) or the recombinant protein is produced in vivo in a GMO and, after binding to the cellular DNA, released by cell disruption. The protein and the binding DNA are then reversibly cross-linked with formaldehyde . By sonication with ultrasound or by digestion with a DNase from Micrococcus sp. the DNA is fragmented. The cross-linked protein-DNA complexes are then immunoprecipitated with an antibody against the recombinant protein or its protein tag . The DNA is released from the complexes by heating and hybridized with the microarray and stained. Finally, the sequences of the hybridized DNA are determined based on their position on the microarray.

One problem with the method in immunoprecipitation is a lack of purity of the DNA-binding protein, which can lead to false positive results. A high specificity of the antibody is also necessary, since heterophile antibodies can also lead to false positive results. In the case of the microarray, insufficient hybridization efficiency can lead to false negative results.

An alternative method is ChIP-Seq , which combines chromatin immunoprecipitation with DNA sequencing at high throughput . Systematic errors in the methods can be partially reduced by using both methods in parallel.

Individual evidence

  1. ^ Y. Blat, N. Kleckner : Cohesins bind to preferential sites along yeast chromosome III, with differential regulation along arms versus the centric region. In: Cell (1999), Volume 98, Issue 2, pp. 249-259. PMID 10428036 .
  2. ↑ MJ Buck, JD Lieb: ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments. In: Genomics (2004), Volume 83, Issue 3, pp. 349-360. PMID 14986705 .
  3. ↑ R. Gottardo: Modeling and analysis of ChIP-chip experiments. In: Methods Mol Biol. (2009), Volume 567, pp. 133-143. doi : 10.1007 / 978-1-60327-414-2_9 . PMID 19588090 .
  4. ↑ CS Pareek, R. Smoczynski, A. Tretyn: Sequencing technologies and genome sequencing. In: J Appl Genet. (2011), Volume 52, Issue 4, pp. 413-435. doi: 10.1007 / s13353-011-0057-x . PMID 21698376 ; PMC 3189340 (free full text).