Immunoprecipitated Chromatin

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Selected Protocol from "Proteins and Proteomics: A Laboratory Manual,", David R. Goodlett, Eugene C. Yi, and Philippe Mottay.

View the protocol "PCR-Based Analysis of Immunoprecipitated Chromatin" at BioSupplynet.com

Alexandre Wagschal, Katia Delaval, Maëlle Pannetier, Philippe Arnaud, and Robert Feil - Institute of Molecular Genetics, CNRS and University of Montpellier, 34293 Montpellier, France

ABSTRACT
After chromatin immunoprecipitation (ChIP), different PCR-based approaches can be used to determine how much DNA is precipitated at a locus of interest. Real-time PCR amplification is often the preferred technique. One can also use duplex PCR amplification, which is the coamplification of a fragment from the region of interest and a control fragment (e.g., the actin gene, or the tubulin gene). This approach allows for estimating relative levels of specific histone modifications along chromosomal domains. For allele-specific studies (for instance, on dosage-compensation mechanisms or on genomic imprinting), electrophoretic detection of single-strand conformation polymorphisms (SSCP) or similar strategies such as hot-stop PCR can differentiate PCR products that represent the silent allele from those amplified from the active allele. If a polymorphic restriction site is present in one allele and absent in the other, the method of choice is hot-stop PCR. If no polymorphic restriction sites are available, but there are single nucleotide polymorphisms (SNPs) that distinguish the alleles of interest, the best approach is to separate the PCR products derived from the two different alleles using SSCP. In SSCP, it is possible to discriminate denatured PCR products derived from one allele or the other because the secondary structure of each single strand will be directly dependent on the sequence itself. Hence, in nondenaturing gel conditions, each single strand will migrate differently. These four PCR-based methodologies to analyze immunoprecipitated chromatin (real-time PCR, duplex PCR, hot-stop PCR, and SSCP) are presented here.