There are mainly two types of ChIP, primarily differing in the starting chromatin preparation. The first uses reversibly
cross-linked chromatin sheared by
sonication called cross-linked ChIP (XChIP). Native ChIP (NChIP) uses native chromatin sheared by
micrococcal nuclease digestion.
Cross-linked ChIP (XChIP) Cross-linked ChIP is mainly suited for mapping the DNA target of transcription factors or other chromatin-associated proteins, and uses reversibly
cross-linked chromatin as starting material. The agent for reversible cross-linking could be
formaldehyde or
UV light. Then the cross-linked chromatin is usually sheared by sonication, providing fragments of 300–1000
base pairs (bp) in length. Mild formaldehyde crosslinking followed by nuclease digestion has been used to shear the chromatin. Chromatin fragments of 400–500 bp have proven to be suitable for ChIP assays as they cover two to three
nucleosomes. Cell debris in the sheared lysate is then cleared by sedimentation and protein–DNA complexes are selectively immunoprecipitated using specific
antibodies to the protein(s) of interest. The antibodies are commonly coupled to
agarose,
sepharose, or magnetic beads. Alternatively, chromatin-antibody complexes can be selectively retained and eluted by inert polymer discs. The immunoprecipitated complexes (i.e., the bead–antibody–protein–target DNA sequence complex) are then collected and washed to remove non-specifically bound chromatin, the protein–DNA
cross-link is reversed and proteins are removed by digestion with
proteinase K. An
epitope-tagged version of the protein of interest, or
in vivo biotinylation can be used instead of antibodies to the native protein of interest. The DNA associated with the complex is then purified and identified by
polymerase chain reaction (PCR),
microarrays (
ChIP-on-chip), molecular cloning and sequencing, or direct high-throughput sequencing (
ChIP-Seq).
Native ChIP (NChIP) Native ChIP is mainly suited for mapping the DNA target of
histone modifiers. Generally, native chromatin is used as starting chromatin. As histones wrap around DNA to form nucleosomes, they are naturally linked. Then the chromatin is sheared by micrococcal nuclease digestion, which cuts DNA at the length of the linker, leaving nucleosomes intact and providing DNA fragments of one nucleosome (200 bp) to five nucleosomes (1000 bp) in length. Thereafter, methods similar to XChIP are used for clearing the cell debris, immunoprecipitating the protein of interest, removing protein from the immunoprecipitated complex, and purifying and analyzing the complex-associated DNA.
Comparison of XChIP and NChIP The major advantage of NChIP is
antibody specificity. Most antibodies to modified histones are raised against unfixed, synthetic peptide antigens. The
epitopes they need to recognize in the XChIP may be disrupted or destroyed by formaldehyde
cross-linking, particularly as the
cross-links are likely to involve
lysine e-amino groups in the N-terminals, disrupting the epitopes. This is likely to explain the consistently low efficiency of XChIP protocols compared to NChIP. But XChIP and NChIP have different aims and advantages relative to each other. XChIP is for mapping target sites of transcription factors and other chromatin-associated proteins; NChIP is for mapping target sites of histone modifiers (see Table 1).
Comparison of ChIP-seq and ChIP-chip Chromatin Immunoprecipitation sequencing, also known as
ChIP-seq, is an experimental technique used to identify transcription factor binding events throughout an entire
genome. Knowing how the proteins in the human body interact with DNA to regulate gene expression is a key component of our knowledge of human diseases and biological processes.
ChIP-seq is the primary technique to complete this task, as it has proven to be extremely effective in resolving how proteins and transcription factors influence phenotypical mechanisms. Overall ChIP-seq has risen to be a very efficient method for determining these factors, but there is a rivaling method known as ChIP-on-chip.
ChIP-on-chip, also known as ChIP-chip, is an experimental technique used to isolate and identify genomic sites occupied by specific DNA-binding proteins in living cells. ChIP-on-chip is a relatively newer technique, as it was introduced in 2001 by Peggy Farnham and Michael Zhang. ChIP-on-chip gets its name by combining the methods of
Chromatin Immunoprecipitation and
DNA microarray, thus creating ChIP-on-chip. The two methods seek similar results, as they both strive to find protein binding sites that can help identify elements in the human genome. Those elements in the human genome are important for the advancement of knowledge in human diseases and biological processes. The difference between ChIP-seq and ChIP-chip is established by the specific site of the protein binding identification. The main difference comes from the efficacy of the two techniques, ChIP-seq produces results with higher sensitivity and spatial resolution because of the wide range of genomic coverage. Even though ChIP-seq has proven to be more efficient than ChIP-chip, ChIP-seq is not always the first choice for scientists. The cost and accessibility of ChIP-seq is a major disadvantage, which has led to the more predominant use of ChIP-chip in laboratories across the world.
Table 1 Advantages and disadvantages of NChIP and XChIP ==History and New ChIP methods==