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59-671: Briefly, the conventional method is as follows: 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 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

118-428: A fluorescent tag such as Cy5 or Alexa 647. Finally, the fragments are poured over the surface of the DNA microarray, which is spotted with short, single-stranded sequences that cover the genomic portion of interest. Whenever a labeled fragment "finds" a complementary fragment on the array, they will hybridize and form again a double-stranded DNA fragment. After a sufficiently large time frame to allow hybridization,

177-423: A permanent wave to hair involves the breaking and reformation of disulfide bonds. Typically a mercaptan such as ammonium thioglycolate is used for the breaking. Following this, the hair is curled and then "neutralized". The neutralizer is typically an acidic solution of hydrogen peroxide, which causes new disulfide bonds to form, thus permanently fixing the hair into its new configuration. Compromised collagen in

236-478: A c-Myc tagging system in yeast. The first demonstration of the mammalian ChIp-on-chip technique reported the isolation of nine chromatin fragments containing weak and strong E2F binding site was done by Peggy Farnham's lab in collaboration with Michael Zhang's lab and published in 2001. This study was followed several months later in a collaboration between the Young lab with the laboratory of Brian Dynlacht which used

295-677: A much wider range of properties than conventional cross-linked elastomers because the domains that act as cross-links are reversible, so can be reformed by heat. The stabilizing domains may be non-crystalline (as in styrene-butadiene block copolymers) or crystalline as in thermoplastic copolyesters. Alkyd enamels , the dominant type of commercial oil-based paint, cure by oxidative crosslinking after exposure to air. In contrast to chemical cross-links, physical cross-links are formed by weaker interactions. For example, sodium alginate gels upon exposure to calcium ions, which form ionic bonds that bridge between alginate chains. Polyvinyl alcohol gels upon

354-459: A polymer depend strongly on the cross-link density. Low cross-link densities increase the viscosities of polymer melts . Intermediate cross-link densities transform gummy polymers into materials that have elastomeric properties and potentially high strengths. Very high cross-link densities can cause materials to become very rigid or glassy, such as phenol-formaldehyde materials. In one implementation, unpolymerized or partially polymerized resin

413-488: A powerful tool in determining genome-wide maps of histone modifications and many more transcription factors. ChIP-on-chip in mammalian systems has been difficult due to the large and repetitive genomes. Thus, many studies in mammalian cells have focused on select promoter regions that are predicted to bind transcription factors and have not analyzed the entire genome. However, whole mammalian genome arrays have recently become commercially available from companies like Nimblegen. In

472-461: A technique developed for large-scale, de novo analysis of higher-order chromatin structures. Cross-link In chemistry and biology , a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural polymers (such as proteins ). In polymer chemistry "cross-linking" usually refers to

531-434: 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

590-399: Is DamID . Also available are antibodies against a specific histone modification like H3 tri methyl K4. As mentioned before, the combination of these antibodies and ChIP-on-chip has become extremely powerful in determining whole genome analysis of histone modification patterns and will contribute tremendously to our understanding of the histone code and epigenetics. A study demonstrating

649-471: Is a highly crosslinked polymer that comprises the main structural material of higher plants. A hydrophobic material, it is derived from precursor monolignols . Heterogeneity arises from the diversity and degree of crosslinking between these lignols. Intrastrand DNA crosslinks have strong effects on organisms because these lesions interfere with transcription and replication . These effects can be put to good use (addressing cancer) or they can be lethal to

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708-683: 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 Better chromatin and protein revery efficiency due to better antibody specificity In 1984 John T. Lis and David Gilmour, at the time a graduate student in the Lis lab, used UV irradiation, a zero-length protein-nucleic acid crosslinking agent, to covalently cross-link proteins bound to DNA in living bacterial cells. Following lysis of cross-linked cells and immunoprecipitation of bacterial RNA polymerase, DNA associated with enriched RNA polymerase

767-509: Is accepted, and which trait for the data is emphasized during the computation. In the recent past, the sliding-window approach seems to be favored and is often described as most powerful. In the third step, these regions are analyzed further. If, for example, the POI was a transcription factor, such regions would represent its binding sites. Subsequent analysis then may want to infer nucleotide motifs and other patterns to allow functional annotation of

826-503: 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

885-549: Is believed that the distribution of modifications and their localizations may offer new insights into the mechanisms of regulation . One of the long-term goals ChIP-on-chip was designed for is to establish a catalogue of (selected) organisms that lists all protein-DNA interactions under various physiological conditions. This knowledge would ultimately help in the understanding of the machinery behind gene regulation, cell proliferation , and disease progression. Hence, ChIP-on-chip offers both potential to complement our knowledge about

944-550: Is commonly used to prepare antibody-hapten conjugates for antibody development. An in-vitro cross-linking method is PICUP ( photo-induced cross-linking of unmodified proteins ). Typical reagents are ammonium persulfate (APS), an electron acceptor, the photosensitizer tris-bipyridylruthenium (II) cation ( [Ru(bpy) 3 ] ). In in-vivo crosslinking of protein complexes, cells are grown with photoreactive diazirine analogs to leucine and methionine , which are incorporated into proteins. Upon exposure to ultraviolet light,

1003-427: Is demonstrated to successfully immunoprecipitate cross-linked chromatin , it is termed " ChIP-grade ". Companies that provide ChIP-grade antibodies include Abcam , Cell Signaling Technology , Santa Cruz, and Upstate. To overcome the problem of specificity, the protein of interest can be fused to a tag like FLAG or HA that are recognized by antibodies. An alternative to ChIP-on-chip that does not require antibodies

1062-426: 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

1121-491: 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 (200bp) to five nucleosomes (1000bp) in length. Thereafter, methods similar to XChIP are used for clearing

1180-462: 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

1239-484: Is to locate protein binding sites that may help identify functional elements in the genome . For example, in the case of a transcription factor as a protein of interest, one can determine its transcription factor binding sites throughout the genome. Other proteins allow the identification of promoter regions , enhancers , repressors and silencing elements , insulators , boundary elements, and sequences that control DNA replication. If histones are subject of interest, it

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1298-780: Is treated with a crosslinking reagent . In vulcanization , sulfur is the cross-linking agent. Its introduction changes rubber to a more rigid, durable material associated with car and bike tires . This process is often called sulfur curing. In most cases, cross-linking is irreversible, and the resulting thermosetting material will degrade or burn if heated, without melting. Chemical covalent cross-links are stable mechanically and thermally. Therefore, cross-linked products like car tires cannot be recycled easily. A class of polymers known as thermoplastic elastomers rely on physical cross-links in their microstructure to achieve stability, and are widely used in non-tire applications, such as snowmobile tracks, and catheters for medical use. They offer

1357-656: 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). Chromatin Immunoprecipitation sequencing, also known as ChIP-seq ,

1416-615: The native state . Common crosslinkers include the imidoester crosslinker dimethyl suberimidate, the N-Hydroxysuccinimide -ester crosslinker BS3 and formaldehyde . Each of these crosslinkers induces nucleophilic attack of the amino group of lysine and subsequent covalent bonding via the crosslinker. The zero-length carbodiimide crosslinker EDC functions by converting carboxyls into amine-reactive isourea intermediates that bind to lysine residues or other available primary amines. SMCC or its water-soluble analog, Sulfo-SMCC,

1475-465: The ChIP-on-chip technique to show for the first time that E2F targets encode components of the DNA damage checkpoint and repair pathways, as well as factors involved in chromatin assembly/condensation, chromosome segregation, and the mitotic spindle checkpoint Other applications for ChIP-on-chip include DNA replication , recombination , and chromatin structure. Since then, ChIP-on-chip has become

1534-517: The DNA microarrays is often a limiting factor to whether a laboratory should proceed with a ChIP-on-chip experiment. Another limitation is the size of DNA fragments that can be achieved. Most ChIP-on-chip protocols utilize sonication as a method of breaking up DNA into small pieces. However, sonication is limited to a minimal fragment size of 200 bp. For higher resolution maps, this limitation should be overcome to achieve smaller fragments, preferably to single nucleosome resolution. As mentioned previously,

1593-558: The DNA sequences used as probes have no fixed distances in the genome. Tiled arrays, however, select a genomic region (or even a whole genome) and divide it into equal chunks. Such a region is called tiled path. The average distance between each pair of neighboring chunks (measured from the center of each chunk) gives the resolution of the tiled path. A path can be overlapping, end-to-end or spaced. Array size : The first microarrays used for ChIP-on-Chip contained about 13,000 spotted DNA segments representing all ORFs and intergenic regions from

1652-450: The ORFs of the genome (that nevertheless remains incomplete, missing intergenic regions) was then applied successfully in three papers published in 2000 and 2001. The authors identified binding sites for individual transcription factors in the budding yeast Saccharomyces cerevisiae . In 2002, Richard Young's group determined the genome-wide positions of 106 transcription factors using

1711-403: The POI form a POI-DNA complex. In the next step, only these complexes are filtered out of the set of DNA fragments, using an antibody specific to the POI. The antibodies may be attached to a solid surface, may have a magnetic bead, or some other physical property that allows separation of cross-linked complexes and unbound fragments. This procedure is essentially an immunoprecipitation (IP) of

1770-442: The actual microarray, other hard- and software equipment is necessary to run ChIP-on-chip experiments. It is generally the case that one company's microarrays can not be analyzed by another company's processing hardware. Hence, buying an array requires also buying the associated workflow equipment. The most important elements are, among others, hybridization ovens, chip scanners, and software packages for subsequent numerical analysis of

1829-399: The addition of borax through hydrogen bonding between boric acid and the polymer's alcohol groups. Other examples of materials which form physically cross-linked gels include gelatin , collagen , agarose , and agar agar . Crosslinking is often measured by swelling tests. The crosslinked sample is placed into a good solvent at a specific temperature, and either the change in mass or

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1888-423: The array is illuminated with fluorescent light. Those probes on the array that are hybridized to one of the labeled fragments emit a light signal that is captured by a camera. This image contains all raw data for the remaining part of the workflow. This raw data, encoded as false-color image , needs to be converted to numerical values before the actual analysis can be done. The analysis and information extraction of

1947-400: The biological question that the experiment seeks to address. Furthermore, due to the different array platforms and lack of standardization between them, data storage and exchange is a huge problem. Generally speaking, the data analysis can be divided into three major steps: During the first step, the captured fluorescence signals from the array are normalized, using control signals derived from

2006-504: The cell debris, immunoprecipitating the protein of interest, removing protein from the immunoprecipitated complex, and purifying and analyzing the complex-associated DNA. 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

2065-531: The change in volume is measured. The more crosslinking, the less swelling is attainable. Based on the degree of swelling, the Flory Interaction Parameter (which relates the solvent interaction with the sample), and the density of the solvent, the theoretical degree of crosslinking can be calculated according to Flory's Network Theory. Two ASTM standards are commonly used to describe the degree of crosslinking in thermoplastics. In ASTM D2765,

2124-552: The cornea, a condition known as keratoconus , can be treated with clinical crosslinking. In biological context crosslinking could play a role in atherosclerosis through advanced glycation end-products (AGEs), which have been implicated to induce crosslinking of collagen, which may lead to vascular stiffening. Proteins can also be cross-linked artificially using small-molecule crosslinkers. This approach has been used to elucidate protein–protein interactions . Crosslinkers bind only surface residues in relatively close proximity in

2183-505: 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 - 500bp 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

2242-414: The crosslinking agents vary greatly. Crosslinking generally involves covalent bonds that join two polymer chains. The term curing refers to the crosslinking of thermosetting resins, such as unsaturated polyester and epoxy resin, and the term vulcanization is characteristically used for rubbers . When polymer chains are crosslinked, the material becomes more rigid. The mechanical properties of

2301-470: The cycle can start again. In the first step, the protein of interest (POI) is cross-linked with the DNA site it binds to in an in vitro environment. Usually this is done by a gentle formaldehyde fixation that is reversible with heat. Then, the cells are lysed and the DNA is sheared by sonication or using micrococcal nuclease . This results in double-stranded chunks of DNA fragments, normally 1 kb or less in length. Those that were cross-linked to

2360-464: The diazirines are activated and bind to interacting proteins that are within a few ångströms of the photo-reactive amino acid analog (UV cross-linking). ChIP-on-chip ChIP-on-chip (also known as ChIP-chip ) is a technology that combines chromatin immunoprecipitation ('ChIP') with DNA microarray ( "chip" ). Like regular ChIP , ChIP-on-chip is used to investigate interactions between proteins and DNA in vivo . Specifically, it allows

2419-845: The distribution of histone H4 on heat shock genes using formaldehyde cross-linking. This technique was extensively developed and refined thereafter. NChIP approach was first described by Hebbes et al ., 1988, and has also been developed and refined quickly. The typical ChIP assay usually takes 4–5 days and requires 10~ 10 cells at least. Now new techniques on ChIP could be achieved as few as 100~1000 cells and completed within one day. ChIP has also been applied for genome-wide analysis by combining with microarray technology ( ChIP-on-chip ) or second-generation DNA-sequencing technology ( Chip-Sequencing ). ChIP can also combine with paired-end tags sequencing in Chromatin Interaction Analysis using Paired End Tag sequencing (ChIA-PET),

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2478-498: The future, as ChIP-on-chip arrays become more and more advanced, high resolution whole genome maps of DNA-binding proteins and chromatin components for mammals will be analyzed in more detail. Introduced in 2007, ChIP sequencing (ChIP-seq) is a technology that uses chromatin immunoprecipitation to crosslink the proteins of interest to the DNA but then instead of using a micro-array, it uses the more accurate, higher throughput method of sequencing to localize interaction points. DamID

2537-471: The genome. Using tiled arrays , ChIP -on-chip allows for high resolution of genome-wide maps. These maps can determine the binding sites of many DNA-binding proteins like transcription factors and also chromatin modifications. Although ChIP-on-chip can be a powerful technique in the area of genomics, it is very expensive. Most published studies using ChIP-on-chip repeat their experiments at least three times to ensure biologically meaningful maps. The cost of

2596-451: The host organism. The drug cisplatin functions by formation of intrastrand crosslinks in DNA. Other crosslinking agents include mustard gas , mitomycin , and psoralen . In proteins , crosslinks are important in generating mechanically stable structures such as hair and wool , skin , and cartilage . Disulfide bonds are common crosslinks. Isopeptide bond formation is another type of protein crosslink. The process of applying

2655-590: The identification of the cistrome , the sum of binding sites , for DNA-binding proteins on a genome-wide basis. Whole-genome analysis can be performed to determine the locations of binding sites for almost any protein of interest. As the name of the technique suggests, such proteins are generally those operating in the context of chromatin . The most prominent representatives of this class are transcription factors , replication -related proteins, like origin recognition complex protein (ORC), histones , their variants, and histone modifications. The goal of ChIP-on-chip

2714-412: 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

2773-567: The non-specific nature of DNA binding proteins has been published in PLoS Biology. This indicates that alternate confirmation of functional relevancy is a necessary step in any ChIP-chip experiment. A first ChIP-on-chip experiment was performed in 1999 to analyze the distribution of cohesin along budding yeast chromosome III. Although the genome was not completely represented, the protocol in this study remains equivalent as those used in later studies. The ChIP-on-chip technique using all of

2832-1014: The orchestration of the genome on the nucleotide level and information on higher levels of information and regulation as it is propagated by research on epigenetics . The technical platforms to conduct ChIP-on-chip experiments are DNA microarrays , or "chips" . They can be classified and distinguished according to various characteristics: Probe type : DNA arrays can comprise either mechanically spotted cDNAs or PCR-products , mechanically spotted oligonucleotides , or oligonucleotides that are synthesized in situ . The early versions of microarrays were designed to detect RNAs from expressed genomic regions ( open reading frames aka ORFs). Although such arrays are perfectly suited to study gene expression profiles , they have limited importance in ChIP experiments since most "interesting" proteins with respect to this technique bind in intergenic regions . Nowadays, even custom-made arrays can be designed and fine-tuned to match

2891-413: 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

2950-436: The protein. This can be done either by using a tagged protein with an antibody against the tag (ex. FLAG , HA , c-myc) or with an antibody to the native protein. The cross-linking of POI-DNA complexes is reversed (usually by heating) and the DNA strands are purified. For the rest of the workflow, the POI is no longer necessary. After an amplification and denaturation step, the single-stranded DNA fragments are labeled with

3009-410: The raw data often remains the most challenging part for ChIP-on-chip experiments. Problems arise throughout this portion of the workflow, ranging from the initial chip read-out, to suitable methods to subtract background noise, and finally to appropriate algorithms that normalize the data and make it available for subsequent statistical analysis , which then hopefully lead to a better understanding of

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3068-421: The raw data. Starting with a biological question, a ChIP-on-chip experiment can be divided into three major steps: The first is to set up and design the experiment by selecting the appropriate array and probe type. Second, the actual experiment is performed in the wet-lab. Last, during the dry-lab portion of the cycle, gathered data are analyzed to either answer the initial question or lead to new questions so that

3127-463: The requirements of an experiment. Also, any sequence of nucleotides can be synthesized to cover genic as well as intergenic regions. Probe size : Early version of cDNA arrays had a probe length of about 200bp. Latest array versions use oligos as short as 70- (Microarrays, Inc.) to 25-mers ( Affymetrix ). (Feb 2007) Probe composition : There are tiled and non-tiled DNA arrays. Non-tiled arrays use probes selected according to non-spatial criteria, i.e.,

3186-505: The same or a second chip. Such control signals tell which probes on the array were hybridized correctly and which bound nonspecifically. In the second step, numerical and statistical tests are applied to control data and IP fraction data to identify POI-enriched regions along the genome. The following three methods are used widely: median percentile rank , single-array error , and sliding-window . These methods generally differ in how low-intensity signals are handled, how much background noise

3245-417: The sample is weighed, then placed in a solvent for 24 hours, weighed again while swollen, then dried and weighed a final time. The degree of swelling and the soluble portion can be calculated. In another ASTM standard, F2214, the sample is placed in an instrument that measures the height change in the sample, allowing the user to measure the volume change. The crosslink density can then be calculated. Lignin

3304-698: The statistical analysis of the huge amount of data generated from arrays is a challenge and normalization procedures should aim to minimize artifacts and determine what is really biologically significant. So far, application to mammalian genomes has been a major limitation, for example, due to the significant percentage of the genome that is occupied by repeats. However, as ChIP-on-chip technology advances, high resolution whole mammalian genome maps should become achievable. Antibodies used for ChIP -on-chip can be an important limiting factor. ChIP -on-chip requires highly specific antibodies that must recognize its epitope in free solution and also under fixed conditions. If it

3363-433: The use of cross-links to promote a change in the polymers' physical properties. When "crosslinking" is used in the biological field, it refers to the use of a probe to link proteins together to check for protein–protein interactions , as well as other creative cross-linking methodologies. Although the term is used to refer to the "linking of polymer chains" for both sciences, the extent of crosslinking and specificities of

3422-408: The yeast genome. Nowadays, Affymetrix offers whole-genome tiled yeast arrays with a resolution of 5bp (all in all 3.2 million probes). Tiled arrays for the human genome become more and more powerful, too. Just to name one example, Affymetrix offers a set of seven arrays with about 90 million probes, spanning the complete non-repetitive part of the human genome with about 35bp spacing. (Feb 2007) Besides

3481-488: Was hybridized to probes corresponding to different regions of known genes to determine the in vivo distribution and density of RNA polymerase at these genes. A year later they used the same methodology to study the distribution of eukaryotic RNA polymerase II on fruit fly heat shock genes. These reports are considered the pioneering studies in the field of chromatin immunoprecipitation. XChIP was further modified and developed by Alexander Varshavsky and co-workers, who examined

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