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Related Concept Videos

Chromatin Immunoprecipitation- ChIP02:36

Chromatin Immunoprecipitation- ChIP

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Chromatin immunoprecipitation, or ChIP, is an antibody-based technique used to identify sites on DNA that bind to transcription factors of interest or histone proteins. It also helps determine the type of histone modifications such as acetylation, phosphorylation, or methylation.
Types of ChIP
ChIP can be divided into two types - X-ChIP and N-ChIP. X-ChIP involves in vivo cross-linking of histones and regulatory proteins to DNA, fragmenting the DNA by sonication, and isolating the protein-DNA...
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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
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Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
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The conversion of alkenes to macromolecules called polymers is a reaction of high commercial importance. The structure of the polymer is defined by a repeating unit, while the terminal groups are considered insignificant. The average degree of polymerization represents the number of repeating units in the polymer molecule and is denoted by the subscript n.
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RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action.
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Proteins that regulate transcription can do so either via direct contact with RNA Polymerase or through indirect interactions facilitated by adaptors, mediators, histone-modifying proteins, and nucleosome remodelers. Direct interactions to activate transcription is seen in bacteria as well as in some eukaryotic genes. In these cases, upstream activation sequences are adjacent to the promoters, and the activator proteins interact directly with the transcriptional machinery. For example, in...
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ChIP-Quantitative Polymerase Chain Reaction (ChIP-qPCR).

Tae Hoon Kim, Job Dekker

    Cold Spring Harbor Protocols
    |May 3, 2018
    PubMed
    Summary

    Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) verifies DNA enrichment for target proteins. This method efficiently analyzes gene regulatory regions across diverse experimental conditions and cell types.

    Area of Science:

    • Molecular Biology
    • Genomics
    • Biochemistry

    Background:

    • Validating target protein-DNA enrichment is crucial in chromatin immunoprecipitation (ChIP) assays.
    • Quantitative PCR (qPCR) offers a sensitive method for detecting DNA sequence enrichment.

    Purpose of the Study:

    • To outline the utility of ChIP-qPCR for confirming specific DNA enrichment by target proteins.
    • To describe strategies for designing qPCR primers for known and candidate genomic binding sites.
    • To highlight ChIP-qPCR as an efficient method for interrogating regulatory regions.

    Main Methods:

    • Designing primers for quantitative PCR (qPCR) targeting known genomic binding sites.
    • Developing qPCR primers for candidate target genes, including promoters and conserved noncoding sequences.

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  • Considering ChIP-chip or ChIP-seq when known sites or candidate genes are unavailable.

    • Main Results:

    • ChIP-qPCR allows for the specific enrichment of DNA sequences associated with target proteins.
    • Primer design enables interrogation of both known and potential regulatory regions.
    • The method is adaptable for various experimental scales (96- or 384-well formats).

    Conclusions:

    • ChIP-qPCR is a cost-effective and efficient strategy for validating ChIP enrichment.
    • It facilitates the analysis of target genes and regulatory elements across numerous conditions and cell types.
    • This technique enhances the reliability of ChIP-based studies in molecular biology and genomics.