ChIP-Seq

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ChIP-Seq flow chart

The ChIP-Seq (from English Chromatin ImmunoPrecipitation DNA-Sequencing ) is a biochemical method for the determination of protein-DNA interactions . The ChIP-Seq is a combination of chromatin immunoprecipitation and DNA sequencing in high throughput . DNA-protein interactions occur in binding sequences for transcription factors on promoters , enhancers , repressors , silencers , isolators and in binding sequences for DNA replication .

properties

The purified recombinant protein is mixed with the DNA or bound in vivo , reversibly cross-linked with formaldehyde . This is followed by fragmentation by ultrasound and immunoprecipitation of the cross-linked protein-DNA complexes with an antibody against the recombinant protein or its protein tag . Finally, the DNA is thermally released and DNA sequencing at high throughput.

An alternative method is the ChIP-on-Chip , which combines chromatin immunoprecipitation with hybridization with a DNA microarray (synonym DNA chip ). Systematic errors in the methods can be partially reduced by using both methods in parallel. In contrast to the ChIP-on-Chip, the ChIP-Seq increases the costs when the sensitivity is increased , since this requires a large number of sequencing processes. A variant of the ChIP-Seq is the Competition ChIP-Seq , which is used to examine whether two transcription factors compete for binding to DNA sequences.

Related methods without immunoprecipitation are the DNAse-Seq and the FAIRE-Seq (formaldehyde-assisted isolation of regulatory elements with high-throughput sequencing), which sequence unfolded regions of the DNA where regulation of gene expression takes place. The ChIRP-Seq is a ChIP-Seq in which DNA-binding RNA can be selectively detected.

The combination of a nuclease protection assay with a ChIP-Seq is called ChIP-Exo , in which DNA areas uncovered by proteins are digested by an exonuclease from bacteriophage λ , while the rest are sequenced.

Individual evidence

  1. ↑ TS Furey: ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions. In: Nat Rev Genet. (2012), Volume 13, Issue 12, pp. 840-852. doi : 10.1038 / nrg3306 . PMID 23090257 ; PMC 3591838 (free full text).
  2. ↑ 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).
  3. ↑ 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 .
  4. ↑ CR Lickwar, F. Mueller, JD Lieb: Genome-wide measurement of protein-DNA binding dynamics using competition ChIP. In: Nature protocols. Volume 8, number 7, 2013, pp. 1337-1353, doi : 10.1038 / nprot.2013.077 , PMID 23764940 .
  5. ↑ L. Song, Z. Zhang, LL Grasfeder, AP Boyle, PG Giresi, BK Lee, NC Sheffield, S. Graef, M. Huss, D. Keefe, Z. Liu, D. London, RM McDaniell, Y. Shibata , KA Showers, JM Simon, T. Vales, T. Wang, D. Winter, Z. Zhang, ND Clarke, E. Birney, VR Iyer, GE Crawford, JD Lieb, TS Furey: Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity. In: Genome research. Volume 21, number 10, October 2011, pp. 1757–1767, doi : 10.1101 / gr.121541.111 , PMID 21750106 , PMC 3202292 (free full text).
  6. Jump up ↑ Ci Chu, Qu, Kun, Zhong, Franklin L., Artandi, Steven E., Chang, Howard Y .: Genomic Maps of Long Noncoding RNA Occupancy Reveal Principles of RNA-Chromatin Interactions . In: Molecular Cell . August 31, 2011. doi : 10.1016 / j.molcel.2011.08.027 .
  7. ↑ Shaun Mahony, B. Franklin Pugh: Protein – DNA binding in high resolution. In: Critical Reviews in Biochemistry and Molecular Biology. 50, 2015, p. 269, doi : 10.3109 / 10409238.2015.1051505 .