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89-508: The goal of ChIP-on-chip 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

178-477: 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,

267-408: A mouse , the plant Arabidopsis thaliana , the puffer fish , and the bacteria E. coli . In December 2013, scientists first sequenced the entire genome of a Neanderthal , an extinct species of humans . The genome was extracted from the toe bone of a 130,000-year-old Neanderthal found in a Siberian cave . New sequencing technologies, such as massive parallel sequencing have also opened up

356-423: 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

445-430: A big potential to modify the genetic control in a host organism. The movement of TEs is a driving force of genome evolution in eukaryotes because their insertion can disrupt gene functions, homologous recombination between TEs can produce duplications, and TE can shuffle exons and regulatory sequences to new locations. Retrotransposons are found mostly in eukaryotes but not found in prokaryotes. Retrotransposons form

534-506: A defined structure that are able to change their location in the genome. TEs are categorized as either as a mechanism that replicates by copy-and-paste or as a mechanism that can be excised from the genome and inserted at a new location. In the human genome, there are three important classes of TEs that make up more than 45% of the human DNA; these classes are The long interspersed nuclear elements (LINEs), The interspersed nuclear elements (SINEs), and endogenous retroviruses. These elements have

623-618: A genome sequence and aids in navigating around the genome. The Human Genome Project was organized to map and to sequence the human genome . A fundamental step in the project was the release of a detailed genomic map by Jean Weissenbach and his team at the Genoscope in Paris. Reference genome sequences and maps continue to be updated, removing errors and clarifying regions of high allelic complexity. The decreasing cost of genomic mapping has permitted genealogical sites to offer it as

712-534: A large portion of the genomes of many eukaryotes. A retrotransposon is a transposable element that transposes through an RNA intermediate. Retrotransposons are composed of DNA , but are transcribed into RNA for transposition, then the RNA transcript is copied back to DNA formation with the help of a specific enzyme called reverse transcriptase. A retrotransposon that carries reverse transcriptase in its sequence can trigger its own transposition but retrotransposons that lack

801-405: A main driving role to generate genetic novelty and natural genome editing. Works of science fiction illustrate concerns about the availability of genome sequences. Michael Crichton's 1990 novel Jurassic Park and the subsequent film tell the story of a billionaire who creates a theme park of cloned dinosaurs on a remote island, with disastrous outcomes. A geneticist extracts dinosaur DNA from

890-413: A major role in shaping the genome. Duplication may range from extension of short tandem repeats , to duplication of a cluster of genes, and all the way to duplication of entire chromosomes or even entire genomes . Such duplications are probably fundamental to the creation of genetic novelty. Horizontal gene transfer is invoked to explain how there is often an extreme similarity between small portions of

979-467: A major theme of the book. The 1997 film Gattaca is set in a futurist society where genomes of children are engineered to contain the most ideal combination of their parents' traits, and metrics such as risk of heart disease and predicted life expectancy are documented for each person based on their genome. People conceived outside of the eugenics program, known as "In-Valids" suffer discrimination and are relegated to menial occupations. The protagonist of

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1068-677: 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

1157-428: A new site. This cut-and-paste mechanism typically reinserts transposons near their original location (within 100 kb). DNA transposons are found in bacteria and make up 3% of the human genome and 12% of the genome of the roundworm C. elegans . Genome size is the total number of the DNA base pairs in one copy of a haploid genome. Genome size varies widely across species. Invertebrates have small genomes, this

1246-501: A precise definition of "genome." It usually refers to the DNA (or sometimes RNA) molecules that carry the genetic information in an organism but sometimes it is difficult to decide which molecules to include in the definition; for example, bacteria usually have one or two large DNA molecules ( chromosomes ) that contain all of the essential genetic material but they also contain smaller extrachromosomal plasmid molecules that carry important genetic information. The definition of 'genome' that

1335-415: A reference, whereas analyses of coverage depth and mapping topology can provide details regarding structural variations such as chromosomal translocations and segmental duplications. DNA sequences that carry the instructions to make proteins are referred to as coding sequences. The proportion of the genome occupied by coding sequences varies widely. A larger genome does not necessarily contain more genes, and

1424-487: A reverse transcriptase must use reverse transcriptase synthesized by another retrotransposon. Retrotransposons can be transcribed into RNA, which are then duplicated at another site into the genome. Retrotransposons can be divided into long terminal repeats (LTRs) and non-long terminal repeats (Non-LTRs). Long terminal repeats (LTRs) are derived from ancient retroviral infections, so they encode proteins related to retroviral proteins including gag (structural proteins of

1513-651: A service, to the extent that one may submit one's genome to crowdsourced scientific endeavours such as DNA.LAND at the New York Genome Center , an example both of the economies of scale and of citizen science . Viral genomes can be composed of either RNA or DNA. The genomes of RNA viruses can be either single-stranded RNA or double-stranded RNA , and may contain one or more separate RNA molecules (segments: monopartit or multipartit genome). DNA viruses can have either single-stranded or double-stranded genomes. Most DNA virus genomes are composed of

1602-404: A single, linear molecule of DNA, but some are made up of a circular DNA molecule. Prokaryotes and eukaryotes have DNA genomes. Archaea and most bacteria have a single circular chromosome , however, some bacterial species have linear or multiple chromosomes. If the DNA is replicated faster than the bacterial cells divide, multiple copies of the chromosome can be present in a single cell, and if

1691-518: A small mitochondrial genome . Algae and plants also contain chloroplasts with a chloroplast genome. The study of the genome is called genomics . The genomes of many organisms have been sequenced and various regions have been annotated. The Human Genome Project was started in October 1990, and then reported the sequence of the human genome in April 2003, although the initial "finished" sequence

1780-560: A species. Within a species, the vast majority of nucleotides are identical between individuals, but sequencing multiple individuals is necessary to understand the genetic diversity. In 1976, Walter Fiers at the University of Ghent (Belgium) was the first to establish the complete nucleotide sequence of a viral RNA-genome ( Bacteriophage MS2 ). The next year, Fred Sanger completed the first DNA-genome sequence: Phage Φ-X174 , of 5386 base pairs. The first bacterial genome to be sequenced

1869-399: 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

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1958-471: 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

2047-556: 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 is an alternative method that does not require antibodies. ChIP-exo uses exonuclease treatment to achieve up to single base pair resolution. CUT&RUN sequencing uses antibody recognition with targeted enzymatic cleavage to address some technical limitations of ChIP. Genome In

2136-509: 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

2225-485: Is also correlated to a small number of transposable elements. Fish and Amphibians have intermediate-size genomes, and birds have relatively small genomes but it has been suggested that birds lost a substantial portion of their genomes during the phase of transition to flight.  Before this loss, DNA methylation allows the adequate expansion of the genome. In humans, the nuclear genome comprises approximately 3.1 billion nucleotides of DNA, divided into 24 linear molecules,

2314-432: Is another DIRS-like elements belong to Non-LTRs. Non-LTRs are widely spread in eukaryotic genomes. Long interspersed elements (LINEs) encode genes for reverse transcriptase and endonuclease, making them autonomous transposable elements. The human genome has around 500,000 LINEs, taking around 17% of the genome. Short interspersed elements (SINEs) are usually less than 500 base pairs and are non-autonomous, so they rely on

2403-549: 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

2492-444: Is carried in plasmids . For this, the word genome should not be used as a synonym of chromosome . Eukaryotic genomes are composed of one or more linear DNA chromosomes. The number of chromosomes varies widely from Jack jumper ants and an asexual nemotode , which each have only one pair, to a fern species that has 720 pairs. It is surprising the amount of DNA that eukaryotic genomes contain compared to other genomes. The amount

2581-601: Is characteristically used for rubbers . When polymer chains are crosslinked, the material becomes more rigid. The mechanical properties of 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

2670-408: Is commonly used in the scientific literature is usually restricted to the large chromosomal DNA molecules in bacteria. Eukaryotic genomes are even more difficult to define because almost all eukaryotic species contain nuclear chromosomes plus extra DNA molecules in the mitochondria . In addition, algae and plants have chloroplast DNA. Most textbooks make a distinction between the nuclear genome and

2759-550: 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,

ChIP-on-chip - Misplaced Pages Continue

2848-427: 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

2937-564: Is even more than what is necessary for DNA protein-coding and noncoding genes due to the fact that eukaryotic genomes show as much as 64,000-fold variation in their sizes. However, this special characteristic is caused by the presence of repetitive DNA, and transposable elements (TEs). A typical human cell has two copies of each of 22 autosomes , one inherited from each parent, plus two sex chromosomes , making it diploid. Gametes , such as ova, sperm, spores, and pollen, are haploid, meaning they carry only one copy of each chromosome. In addition to

3026-492: Is facilitated by active DNA demethylation , a process that entails the DNA base excision repair pathway. This pathway is employed in the erasure of CpG methylation (5mC) in primordial germ cells. The erasure of 5mC occurs via its conversion to 5-hydroxymethylcytosine (5hmC) driven by high levels of the ten-eleven dioxygenase enzymes TET1 and TET2 . Genomes are more than the sum of an organism's genes and have traits that may be measured and studied without reference to

3115-401: Is rather exceptional, eukaryotes generally have these features in their genes and their genomes contain variable amounts of repetitive DNA. In mammals and plants, the majority of the genome is composed of repetitive DNA. High-throughput technology makes sequencing to assemble new genomes accessible to everyone. Sequence polymorphisms are typically discovered by comparing resequenced isolates to

3204-421: Is to reduce the number of genes in a genome to the bare minimum and still have the organism in question survive. There is experimental work being done on minimal genomes for single cell organisms as well as minimal genomes for multi-cellular organisms (see developmental biology ). The work is both in vivo and in silico . There are many enormous differences in size in genomes, specially mentioned before in

3293-780: 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

3382-498: The University of Hamburg , Germany. The website Oxford Dictionaries and the Online Etymology Dictionary suggest the name is a blend of the words gene and chromosome . However, see omics for a more thorough discussion. A few related -ome words already existed, such as biome and rhizome , forming a vocabulary into which genome fits systematically. It is very difficult to come up with

3471-615: 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,

3560-517: 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,

3649-557: 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

ChIP-on-chip - Misplaced Pages Continue

3738-506: 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 a c-Myc tagging system in yeast. The first demonstration of

3827-403: 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

3916-426: The X and Y chromosomes of mammals, so the technical definition of the genome must include both copies of the sex chromosomes. For example, the standard reference genome of humans consists of one copy of each of the 22 autosomes plus one X chromosome and one Y chromosome. A genome sequence is the complete list of the nucleotides (A, C, G, and T for DNA genomes) that make up all the chromosomes of an individual or

4005-442: 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

4094-399: 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

4183-423: 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

4272-400: 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

4361-412: The blood of ancient mosquitoes and fills in the gaps with DNA from modern species to create several species of dinosaurs. A chaos theorist is asked to give his expert opinion on the safety of engineering an ecosystem with the dinosaurs, and he repeatedly warns that the outcomes of the project will be unpredictable and ultimately uncontrollable. These warnings about the perils of using genomic information are

4450-549: The cells divide faster than the DNA can be replicated, multiple replication of the chromosome is initiated before the division occurs, allowing daughter cells to inherit complete genomes and already partially replicated chromosomes. Most prokaryotes have very little repetitive DNA in their genomes. However, some symbiotic bacteria (e.g. Serratia symbiotica ) have reduced genomes and a high fraction of pseudogenes: only ~40% of their DNA encodes proteins. Some bacteria have auxiliary genetic material, also part of their genome, which

4539-531: 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,

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4628-478: The chromosomes in the nucleus, organelles such as the chloroplasts and mitochondria have their own DNA. Mitochondria are sometimes said to have their own genome often referred to as the " mitochondrial genome ". The DNA found within the chloroplast may be referred to as the " plastome ". Like the bacteria they originated from, mitochondria and chloroplasts have a circular chromosome. Unlike prokaryotes where exon-intron organization of protein coding genes exists but

4717-552: 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

4806-470: 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

4895-445: The details of any particular genes and their products. Researchers compare traits such as karyotype (chromosome number), genome size , gene order, codon usage bias , and GC-content to determine what mechanisms could have produced the great variety of genomes that exist today (for recent overviews, see Brown 2002; Saccone and Pesole 2003; Benfey and Protopapas 2004; Gibson and Muse 2004; Reese 2004; Gregory 2005). Duplications play

4984-518: The fields of molecular biology and genetics , a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses ). The nuclear genome includes protein-coding genes and non-coding genes, other functional regions of the genome such as regulatory sequences (see non-coding DNA ), and often a substantial fraction of junk DNA with no evident function. Almost all eukaryotes have mitochondria and

5073-470: The film is an In-Valid who works to defy the supposed genetic odds and achieve his dream of working as a space navigator. The film warns against a future where genomic information fuels prejudice and extreme class differences between those who can and cannot afford genetically engineered children. 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

5162-534: 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 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

5251-502: 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 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

5340-470: 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

5429-406: The genomes of two organisms that are otherwise very distantly related. Horizontal gene transfer seems to be common among many microbes . Also, eukaryotic cells seem to have experienced a transfer of some genetic material from their chloroplast and mitochondrial genomes to their nuclear chromosomes. Recent empirical data suggest an important role of viruses and sub-viral RNA-networks to represent

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5518-451: 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

5607-455: The human genome All the cells of an organism originate from a single cell, so they are expected to have identical genomes; however, in some cases, differences arise. Both the process of copying DNA during cell division and exposure to environmental mutagens can result in mutations in somatic cells. In some cases, such mutations lead to cancer because they cause cells to divide more quickly and invade surrounding tissues. In certain lymphocytes in

5696-425: The human genome and 9% of the fruit fly genome. Tandem repeats can be functional. For example, telomeres are composed of the tandem repeat TTAGGG in mammals, and they play an important role in protecting the ends of the chromosome. In other cases, expansions in the number of tandem repeats in exons or introns can cause disease . For example, the human gene huntingtin (Htt) typically contains 6–29 tandem repeats of

5785-517: The human immune system, V(D)J recombination generates different genomic sequences such that each cell produces a unique antibody or T cell receptors. During meiosis , diploid cells divide twice to produce haploid germ cells. During this process, recombination results in a reshuffling of the genetic material from homologous chromosomes so each gamete has a unique genome. Genome-wide reprogramming in mouse primordial germ cells involves epigenetic imprint erasure leading to totipotency . Reprogramming

5874-563: 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 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)

5963-517: 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 the ChIP-on-chip technique to show for

6052-476: The multicellular eukaryotic genomes. Much of this is due to the differing abundances of transposable elements, which evolve by creating new copies of themselves in the chromosomes. Eukaryote genomes often contain many thousands of copies of these elements, most of which have acquired mutations that make them defective. Here is a table of some significant or representative genomes. See #See also for lists of sequenced genomes. Initial sequencing and analysis of

6141-565: 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

6230-795: The nucleotides CAG (encoding a polyglutamine tract). An expansion to over 36 repeats results in Huntington's disease , a neurodegenerative disease. Twenty human disorders are known to result from similar tandem repeat expansions in various genes. The mechanism by which proteins with expanded polygulatamine tracts cause death of neurons is not fully understood. One possibility is that the proteins fail to fold properly and avoid degradation, instead accumulating in aggregates that also sequester important transcription factors, thereby altering gene expression. Tandem repeats are usually caused by slippage during replication, unequal crossing-over and gene conversion. Transposable elements (TEs) are sequences of DNA with

6319-1014: 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

6408-447: The organelle (mitochondria and chloroplast) genomes so when they speak of, say, the human genome, they are only referring to the genetic material in the nucleus. This is the most common use of 'genome' in the scientific literature. Most eukaryotes are diploid , meaning that there are two of each chromosome in the nucleus but the 'genome' refers to only one copy of each chromosome. Some eukaryotes have distinctive sex chromosomes, such as

6497-590: The proportion of non-repetitive DNA decreases along with increasing genome size in complex eukaryotes. Noncoding sequences include introns , sequences for non-coding RNAs, regulatory regions, and repetitive DNA. Noncoding sequences make up 98% of the human genome. There are two categories of repetitive DNA in the genome: tandem repeats and interspersed repeats. Short, non-coding sequences that are repeated head-to-tail are called tandem repeats . Microsatellites consisting of 2–5 basepair repeats, while minisatellite repeats are 30–35 bp. Tandem repeats make up about 4% of

6586-412: The prospect of personal genome sequencing as a diagnostic tool, as pioneered by Manteia Predictive Medicine . A major step toward that goal was the completion in 2007 of the full genome of James D. Watson , one of the co-discoverers of the structure of DNA. Whereas a genome sequence lists the order of every DNA base in a genome, a genome map identifies the landmarks. A genome map is less detailed than

6675-436: 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

6764-439: The proteins encoded by LINEs for transposition. The Alu element is the most common SINE found in primates. It is about 350 base pairs and occupies about 11% of the human genome with around 1,500,000 copies. DNA transposons encode a transposase enzyme between inverted terminal repeats. When expressed, the transposase recognizes the terminal inverted repeats that flank the transposon and catalyzes its excision and reinsertion in

6853-410: 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

6942-421: 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

7031-463: 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.,

7120-505: 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

7209-417: 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

7298-423: The shortest 45 000 000 nucleotides in length and the longest 248 000 000 nucleotides, each contained in a different chromosome. There is no clear and consistent correlation between morphological complexity and genome size in either prokaryotes or lower eukaryotes . Genome size is largely a function of the expansion and contraction of repetitive DNA elements. Since genomes are very complex, one research strategy

7387-698: 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

7476-398: The term is used to refer to the "linking of polymer chains" for both sciences, the extent of crosslinking and specificities of 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

7565-541: The virus), pol (reverse transcriptase and integrase), pro (protease), and in some cases env (envelope) genes. These genes are flanked by long repeats at both 5' and 3' ends. It has been reported that LTRs consist of the largest fraction in most plant genome and might account for the huge variation in genome size. Non-long terminal repeats (Non-LTRs) are classified as long interspersed nuclear elements (LINEs), short interspersed nuclear elements (SINEs), and Penelope-like elements (PLEs). In Dictyostelium discoideum , there

7654-407: 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

7743-429: Was completed in 1996, again by The Institute for Genomic Research. The development of new technologies has made genome sequencing dramatically cheaper and easier, and the number of complete genome sequences is growing rapidly. The US National Institutes of Health maintains one of several comprehensive databases of genomic information. Among the thousands of completed genome sequencing projects include those for rice ,

7832-477: Was missing 8% of the genome consisting mostly of repetitive sequences. With advancements in technology that could handle sequencing of the many repetitive sequences found in human DNA that were not fully uncovered by the original Human Genome Project study, scientists reported the first end-to-end human genome sequence in March 2022. The term genome was created in 1920 by Hans Winkler , professor of botany at

7921-400: Was that of Haemophilus influenzae , completed by a team at The Institute for Genomic Research in 1995. A few months later, the first eukaryotic genome was completed, with sequences of the 16 chromosomes of budding yeast Saccharomyces cerevisiae published as the result of a European-led effort begun in the mid-1980s. The first genome sequence for an archaeon , Methanococcus jannaschii ,

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