4023-1 Genome Structure, Organ is at Ions and Methods of Analysis(1)

8/6/2019 4023-1 Genome Structure, Organ is at Ions and Methods of Analysis(1)

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Sugars:Gluconeogenesis: takes place in the liver and to a lesser extent the kidney.

Basic sugar unit is a MONOSACCHARIDE:

These derive their names from the chemical nature of their carbonyl group and the number ofcarbons in the molecule. The smallest monosaccharides have 3 carbons and are called

trioses.A 4 carbon atom sugar is a TETROSE, 5= PENTOSE, 6 = HEXOSE etc etc.

If the carbonyl group is an aldehyde (as in glucose and ribose) the sugar is an ALDOSEIf the carbonyl group is a ketone the sugar is a KETOSE.


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Inosine

Inosine is commonly found in tRNAs and is essential for proper translation of

the genetic code in Watson and Crick wobble base pairing.

7-methylguanosine

A 7-methylguanosine 5' cap is added to precursor messenger RNA and someother primary RNA transcripts found in eukaryotes and, as a specialexception, caliciviruses such as noroviruses. The process of 5' capping is vitalto creating matur messenger RNA which is then able to undergo translation.

5-methylcytosine

Cytosine residues occurring in CpG dinucleotides in vertebrate DNA aretargets for methylation by a specific cytosine methyltransferase. Methylationoccurs at carbon atom 5 of the cytosine to generate 5-methylcytosine, which ischemically unstable and can spontaneously deaminate to give thymine. Overlong periods of evolutionary time, the number of CpGs in vertebrate DNA hasgradually been eroded, although regions of the normal (expected) CpGfrequency are known and often mark transcriptionally active sequences (CpGislands).

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A-form B-form Z-formHelix right-handed right-handed left-handed

Repeating unit 1 bp 1 bp 2 bpMean bp/turn 11 10.5 12Diameter 2.6 nm 2.0 nm 1.8 nm

The double helix described by Watson and Crick is called the B-form of DNA.Its characteristic features lie in its dimensions: a helical diameter of 2.37 nm, arise of 0.34 nm per base pair, and a pitch (i.e. distance taken up by a completeturn of the helix) of 3.4 nm, this corresponding to ten base pairs per turn. TheDNA in living cells is thought to be predominantly in this B-form, but it is now

clear that genomic DNA molecules are not entirely uniform in structure.

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Plasmids are circular, double-stranded DNA (dsDNA) molecules that are under normalconditions replicate and are mainteined as distinct separate moleculares from a cellschromosomal DNA. These extrachromosomal DNAs, which occur naturally in bacteria, yeast,

and some higher eukaryotic cells, exist in a parasitic or symbiotic relationship with their hostcell.

The average number of molecules of a given plasmid per cell is called its copy number. Largeplasmids (>40 kilobase pairs) are often conjugative, have small copy numbers (1 to several percell), code for all functions required for their replication, and partition themselves amongdaughter cells during cell division in a manner similar to the bacterial chromosome.

Plasmids smaller than 7.5 kilobase pairs usually are nonconjugative, have high copy numbers(typically 1020 per cell), rely on their host to provide some functions required for replication,and are distributed randomly between daughter cells at division. Many plasmids that are ableto replicate and be efficiently maintained in eukaryotes require a require stab (stability)

element(s).

Many plasmids control medically important properties of pathogenic bacteria, includingresistance to one or several antibiotics, production of toxins, and synthesis of cell surfacestructures required for adherence or colonization. Plasmids that determine resistance toantibiotics are often called R plasmids (or R factors). Representative toxins encoded byplasmids include heat-labile and heat-stable enterotoxins ofE. coli, exfoliative toxin ofStaphylococcus aureus, and tetanus toxin ofClostridium tetani. Some plasmids are cryptic andhave no recognizable effects on the bacterial cells that harbor them. Comparing plasmidprofiles is a useful method for assessing possible relatedness of individual clinical isolates of aparticular bacterial species for epidemiological studies.

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Genomes can be composed of one or a number of nucleic acid molecules andcan vary greatly in size and structure

Nanoarchaeum equitans: 552 genes. The smallest genome of a true organismyet found. A parasitic member of the archaea bacteria.Mycoplasma genitalium: smallest number of genes 485 protein-encodinggenes. Using this organism The J. Craig Venter Institute concluded only 381genes are essential for life.

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The smallest viruses contain only a few genes and can have an RNA or a DNAgenome; the largest viruses contain hundreds of genes and have a double-

stranded DNA genome. Some examples of these types of viruses are asfollows: single-stranded RNAtobacco mosaic virus, bacteriophage R17,poliovirus; double-stranded RNAreovirus; single-stranded DNAparvovirus;single-stranded circular DNAM13 and fX174 bacteriophages; double-strandedcircular DNASV40 and polyomaviruses; double-stranded DNAT4bacteriophage, herpes virus; double-stranded DNA with covalently linkedterminal proteinadenovirus; double-stranded DNA with covalently sealed endspoxvirus. Thepeculiar ends (as well as the circular forms) overcome the difficulty of

replicating the last few nucleotides at the end of a DNA chain

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Bacterial DNA is freely floating in the cytoplasm. Prokaryotic chromatin is,nevertheless, highly condensed and forms a pseudo-compartment that

frequently occupies a distinct region within the cell, which is characterized bythe absence of ribosomes. This chromatin dense area is the functional (butNOT structural) equivalent to the eukaryotic nucleus and is, therefore, termedthe bacterial nucleoid. The bacterial nucleoid is a complex structure dividedinto different, hierarchic levels of organization. A number of mechanisms foldchromosomal DNA into discrete supercoiled loops that are dynamicallyrearranged to fit the needs of the growing cell. These fluid topological domains,might interact with each other to form condensed filaments and loops, asrecently suggested by atomic force microscopy ofE. colichromatin and finally

assemble into a defined ring-like superstructure. The mechanisms mediatingthe establishment of these distinct patterns are still unclear and theiridentification might necessitate the combination of cell biological and highresolution electron microscopic approaches.

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The human mitochondrial genome

An average human cell contains about 8000 mitochondrial genome copies, 10

or so in each mitochondrionThe human mitochondrial genome contains 37 genes and is small andcompact, with little wasted space, so much so that the ATP6 and ATP8 genesoverlap.

SNPs:Although almost all (99.9%) nucleotide bases are exactly the same inall people, scientists have identified about 1.4 million locations where single-base DNA differences (SNPs) occur in humans. This information promises torevolutionize the processes of finding chromosomal locations for disease-

associated sequences and tracing human history.

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The chromosome content (number of copies of the chromosome per set) isknown as the ploidy number. For humans the haploid ploidy number (n) is 23:

21 autosomes (numbers 1-21) and 2 sex (X,Y) chromosomes.

Most human somatic cells however are diploid 2n (=46), although there areexceptions. Some terminally differentiated cells, such as red blood cells,keratinocytes and platelets, have no nuclei and are described as nulliploid.Other cells are polyploid (exact multiples of n) as a result of DNA replicationwithout cell division (endomitosis), or as a result of cell fusion. For example,the ploidy of hepatocytes ranges from 2n to 8n (8 times the haploid number =184 chromosomes!), that of cardiomyocytes (heart muscle cells) from 4n to 8n,and that of the giant megakaryocytes of the bone marrow from 16n to 64n. The

latter cells individually give rise to thousands of nulliploid platelet cells.Polyploidy also occurs as a result of cell fusion (e.g. muscle fibers) fusemultiple diploid nuclei, to form a multi-nucleated cell (syncytium).

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Satellite DNAThe human genome contains arrays, of highly repetitive DNA Tandem repeatsequences. These may be simple (1-10) or moderately complex (10s-100s)nucleotides in length. Satellite arrays occur at a few specific and/or manydifferent chromosomal locations and have a different density from bulk DNAand therefore form satellite bands when genomic DNA is separated on adensity gradient. When DNA is isolated from human cells by conventionalmethods, it is subject to mechanical shearing. Fragments are generated fromthe bulk DNA (with a base composition of ~42% GC) and fragments from thesatellite DNA regions which may have a similar or different base composition.If the base composition is significantly different, satellite DNA sequences canbe separated from the bulk DNA by buoyant density gradient centrifugation.Following centrifugation, they appear as minor (or satellite) bands of different

buoyant density from a major band which represents bulk DNA (see picture inslide). Typically, human DNA is complexed with Ag+ ions and then fractionatedin buoyant density gradients containing cesium sulfate, whereupon threesatellite bands are identified at different densities: satellite 1 1.687 gcm-3;satellite 2 1.693 gcm-3; satellite 3 1.697 gcm-3

Some satellite DNA sequence cannot easily be resolved by density gradientcentrifugation. These sequences were first identified by digestion of genomicDNA with a restriction endonuclease which typically has a single recognitionsite in the basic repeat unit.

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Satellite DNA is classified into 3 subclasses:

Satellite Minisatellite and Microsatellite DNA.

Each of the satellite sub-classes includes a number of different tandemlyrepeated DNA sequence families, some of which are shared between differentclasses. Some of the repetitive DNA families in the satellites are based on verysimple repeat units. Additional higher order repeat units are superimposed onthe small basic repeat units and are thought to have arisen as a result ofsubsequent amplification of a unit which is larger than the initial basic repeatunit and contains some diverged units.

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Alphoid DNA and centromeric heterochromatinWhilst the exact function(s) of this satellite DNA remains unclear alpha satellite(or alphoid DNA) constitutes the bulk of the centromeric heterochromatin andaccounts for about 35% of the DNA of each chromosome. It is characterizedby tandem repeats of a basic mean length of 171 bp, although higher orderunits are also seen. The sequence divergence between individual members ofthe alphoid DNA family can be so high that it is possible to isolatechromosome-specific subfamilies for each of the human chromosomes. Thecentromeric -satellite repeat units often contain a binding site for a specificcentromereprotein, CENP-B and are involved in centromere formation.

The S.pombe proteins Abp1, Cbh1, and Cbh2 are homologs of the humanCENP-B protein. Conserved heterochromatin-specific modifications of the

histone H3 tail, involving deacetylation of Lys 9 and Lys 14 and subsequentmethylation of Lys 9, promote the recruitment of a heterochromatin protein,Swi6, a homolog of Human heterochromatin protein 1 (HP1). Disruption ofAbp1, Cbh1, and Cbh2 in S.pombe causes a decrease in heterochromatin-specific modifications of histone H3 and in the absence of Abp1 or Cbh1,centromeric association of Swi6 is diminished. These results indicate that theCENP-B homologs act as site-specific nucleation factors for the formation ofcentromeric heterochromatin by heterochromatin-specific modifications ofhistone tails.

http://genesdev.cshlp.org/content/16/14/1766.abstract

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Minisatellites 90% are found at telomeric and sub-telomeric regions. Repeat numbervariations exist at over a 1000 locations.

~1% of minisatellites are hypervariable containing a "core consensus

GGGCAGGANG sequence or more generally a strand bias with purines (A and G) onone strand and pyrimidines (C and T) on the other. This sequence is similar in sizeand in G content to the chisequence, a signal for generalized recombination in E.coli. Indeed hypervariables, have an average mutation rate in the germline 0.5-20%higher than the genome average, making them the most genetically unstableregions in the human genome known to date. The most highly mutable minisatellitelocus described so far is CEB1 (D2S90). Minisatellites are thought to have arisen dueto action of meiotic recombination hotpsots, and/or replication slippage events.

The extreme sequence variation (polymorphism) between minisatellites of individualsmakes them ideal DNA fingerprinting substrates and genetic markers in linkage

analysis and population studies. The great majority of hypervariable minisatellite DNAsequences are not transcribed, except for elements occuring within noncodingintragenic sequences. Some, however, are expressed and been implicated asregulators of gene expression at the levels of transcription, alternative splicing andgenetic imprinting control. For example, the MUC1 locus on 1q is an expressedhypervariable minisatellite locus. It encodes a glycoprotein found in several epithelialtissues and body fluids which is highly polymorphic as a result of extensive variationin the number of minisatellite-encoded repeats. MUC1 is over expressed in manycancers.

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Microsatellites: are examples of tandemly repeated DNA regions thought to becreated by errors in genome replication. The microsatellite repeat unit is short up to12 bp in length. The commonest type of human microsatellite are dinucleotiderepeats, with approximately 140,000 copies in the genome as a whole, about half ofthese being repeats of the motif CA. Single-nucleotide repeats (e.g., AAAAA) arethe next most common (about 120,000 copies in total).

Although their exact function, if any, is unknown, microsatellites have proved veryuseful to geneticists. Many microsatellites are variable, meaning that the number ofrepeat units in the array is different in different members of a species. This is becauseslippage sometimes occurs when a microsatellite is copied during DNA replication,leading to insertion or, less frequently, deletion of one or more of the repeat units. Notwo humans alive today have exactly the same combination of microsatellite lengthvariants: if enough microsatellites are examined then a unique genetic profile can beestablished for every person. The only exceptions are genetically identical twins.Genetic profiling is well known as a tool in forensic science and genetic profilingwhere microsatellites can be used to establish kinship relationships and population

affinities.

The use of microsatellite analysis in genetic profiling.In the example show in the slide, microsatellites located on the short arm ofchromosome 6 have been amplified by PCR. The PCR products are labeled with ablue or green fluorescent marker and run in a polyacrylamide gel, each lane showingthe genetic profile of a different individual. No two individuals have the same geneticprofile because each person has a different set of microsatellite length variants, thevariants giving rise to bands of different sizes after PCR. The red bands are DNA sizemarkers. Image courtesy of Applied Biosystems, Warrington, UK.

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Pseudogenes are defunct relatives of known genes that have lost their coding

ability or are otherwise no longer expressed. A conventional pseudogene is agene that has been inactivated because its nucleotide sequence has changedby mutation. Once a pseudogene has become nonfunctional it will degradethrough accumulation of more mutations, and eventually will no longer berecognizable as a gene relic. The human globin pseudogenes are examples of

conventional pseudogenes.

A processed pseudogene arises not by evolutionary decay but by anabnormal adjunct to gene expression. A processed pseudogene is derivedfrom a reverse transcription of mRNA into a cDNA copy which subsequently

integratates into the same chromosome as its functional parent, or possiblyinto a different chromosome. Because a processed pseudogene is a copy ofan mRNA molecule, it does not contain any of the introns that were present inits parent gene. It also lacks the nucleotide sequences immediately upstream

of the parent gene, which is the region in which the signals used to switch onexpression of the parent gene are located. The absence of these signalsmeans that a processed pseudogene is usually inactive.

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Active protein encoding genes make up only a small part of the human

genome

All the exons of the human genome make up only 48 Mb (1.5%) of the totalDNA content. The average protein gene consists of 3000 bases, but sizes varygreatly, with the largest known human gene being dystrophin at 2.4 millionbases. The total number of genes is estimated at 20,000-25,000 and the

functions of over 50% of discovered genes is not yet known.

Gene fragments are short, isolated exon regions from within a gene. Newstudies in Rice have shown these fragments can become transcriptionallyactive by acquiring a donor gene promoter.

OMIM is a comprehensive, authoritative, and timely compendium of humangenes and genetic phenotypes. The full-text, referenced overviews in OMIMcontain information on all known mendelian disorders and over 12,000 genes.

OMIM focuses on the relationship between phenotype and genotype. It isupdated daily, and the entries contain copious links to other geneticsresources.

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A typical human gene coding chromosomal region

To answer this question examine a 50 kb segment of chromosome 12. Thissegment contains the following genetic features:

a) Four genes. Note that each of these four genes is discontinuous, the

number of introns ranging from two for SYB1 to eight for PKP2.b) 88 genome-wide repeat sequences.c) Seven microsatellites: sequences in which a short DNA motif is repeatedin tandem. One of the microsatellites seen here has the motif CA repeated 12times, giving the sequence:5CACACACACACACACACACACACA3 3GTGTGTGTGTGTGTGTGTGTGTGT5The other six microsatellites comprise repeats of CAAA, CCTG, CTGGGG,CAAAA, TG, and TTTG, respectively. Four of the seven microsatellites arelocated within introns.d) Approximately 30% nongenic, nonrepetitive, single-copy DNA of no

known function or significance.

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Ultraconserved regions (UCRs) of the human genome are almost completelyconserved among various species. The UCRs represent a small fraction of the humangenome that are likely to be functional but not encoding proteins and have beencalled the dark matter of the human genome. Because of the high degree ofconservation, the UCRs may have fundamental functional importance for theontogeny and phylogeny of mammals and other vertebrates.

There are 481 segments longer than 200 base pairs (bp) that are absolutelyconserved (100% identity with no insertions or deletions) between orthologousregions of the human, rat, and mouse genomes. Nearly all of these segments are alsoconserved in the chicken and dog genomes, with an average of 95 and 99% identity,respectively. Many are also significantly conserved in fish. These ultraconservedelements of the human genome are most often located either overlapping exons ingenes involved in RNA processing or in introns or nearby genes involved in theregulation of transcription and development. Many UCRs contain miRNAs and arelocated at chromosomal fragile sites. As well as being expressed in normal tissue

many of the miRNAs are expressed in cancerous cells. For example, the activemolecules of the miR-16-1/miR-15a cluster, shown to be an essential player in theinitiation of chronic lymphocytic leukemia (CLL), are completely conserved in human,mouse, and rat and highly conserved in nine out of the ten sequenced primatespecies.

In addition to the 481 segments longer than 200bp fragments, more than 5000sequences of over 100 bp are absolutely conserved among the three sequencedmammals (humans, mouse and rat). These represent a class of genetic elementswhose functions and evolutionary origins are yet to be determined, but which aremore highly conserved between these species than are proteins and appear to beessential for the ontogeny of mammals and other vertebrates.

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Fluorescent dyes

In vivo dyes

DAPI or4',6-diamidino-2-phenylindole is a fluorescent stain that bindsstrongly to DNA. It is used extensively in fluorescence microscopy. Since DAPIwill pass through an intact cell membrane, it may be used to stain both live andfixed cells.Hoechst 33342: The Hoechst stains are part of a family of fluorescent stainsfor labelling DNA in fluorescence microscopy and fluorescent-activated cellsorting (FACS). Because fluorescent stains label DNA, they are alsocommonly used to visualize nuclei and mitochondria. Fluorescent stains suchas the above can be used to locate the nucleus and track the stage of cell

cycle or DNA content.

In vitro dyes

Ethidium bromide ("EtBr) is an intercalating agent commonly used as anucleic acid stain in techniques such as agarose gel electrophoresis.Commonly abbreviated as when exposed to ultraviolet light, it will fluorescewith an orange colour, intensifying almost 20-fold after binding to DNA.Ethidium bromide may be a mutagen, carcinogen or teratogen although thisdepends on the organism and conditions employed.

SYBR dyes (red green gold)SYBR Green I (SG) is an asymmetrical cyanine dye. When SYBR Green Ibinds to DNA, the resulting DNA-dye-complex absorbs blue light (max = 488nm) and emits green light (max = 522 nm). The stain preferentially binds todouble-stranded DNA, but will stain single-stranded DNA with lower 33


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The preparation and study of karyotypes is part of cytogenetics.

A karyotype describes the number and appearance of the chromosomes

contained within a eukaryote cell nucleus of either an individual organism or aspecies. When defining a karyotype attention is paid to chromosome length,the position of the centromeres, any differences between the sexchromosomes, and any other physical characteristics.

Distinguishing chromosome features revealed when chromosomes arestained to produce a karyogramThere are a number of different staining techniques (Table 7.1), each resultingin a banding pattern that is characteristic for a particular chromosome.

Constitutive heterochromatin domains occur throughout the chromosomesof eukaryotes, but particularly at the centromeres and telomeres. They oftenconsist of very highly condensed, repetitive DNA and are largelytranscriptionally silent.

Where are genes located on chromosomes?The uneven gene distribution within human chromosomes was suspected forseveral years before the sequence was completed. There were two lines ofevidence, one of which related to the banding patterns that are produced whenchromosomes are stained. The dyes used in these procedures (see Table 7.1)

bind to DNA molecules, but in most cases with preferences for certain basepairs. Giemsa, It is a mixture of two chemicals, methylene blue and eosin. Itbinds to the phosphate of DNA and has a greater affinity for DNA regions thatare rich in A and T nucleotides. The dark G-bands in the human karyogram aretherefore thought to be AT-rich regions of the genome. The base compositionof the genome as a whole is 59.7% A + T so the dark G-bands must have AT 34


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The double-helical structure of nucleic acids, in which one strand binds specifically to its exactcomplement, is a fortuitous design for the study of biology. The ease of constructing assays basedon this feature is responsible for the incredibly fast pace that has characterized molecular biologyresearch since its inception. With the incorporation of fluorescence technologies, the ability to design

multiplexed and high-throughput assays has increased the pace still further, to the point in whichsophisticated bioinformatics are required to analyze the huge outpouring of data. From nucleic acidsequencing to real-time polymerase chain reaction (PCR) assays to microarrays, the new genomics eraowes its development in large part to the development of fluorescence methodologies.

Nucleic acid hybridization involves mixing single strands of two sources of nucleic acids, a probe whichtypically consists of a homogeneous population ofidentified molecules (e.g. cloned DNA or chemicallysynthesized oligonucleotides) and a target which typically consists of a complex, heterogeneouspopulation of nucleic acid molecules. If either the probe or the target is initially double-stranded, theindividual strands must be separated, generally by heating or by alkaline treatment. After mixing singlestrands of probe with single strands of target, strands with complementary base sequences can beallowed to reassociate. Complementary probe strands can reanneal to form homoduplexes, as cancomplementary target DNA strands. However, it is the annealing of a probe DNA strand and acomplementary target DNA strand to form a labeled probe-target heteroduplex that defines the

usefulness of a nucleic acid hybridization assay. The rationale of the hybridization assay is to use theidentified probe to query the target DNA by identifying fragments in the complex target which may berelated in sequence to the probe.


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Melting temperature and hybridization stringency

Denaturation of double-stranded probe DNA is generally achieved by heating a

solution of the labeled DNA to a temperature which disrupts the hydrogenbonds that hold the two complementary DNA strands together. The energyrequired to separate two perfectly complementary DNA strands is dependenton a number of factors, notably:strand length - long homoduplexes contain a large number of hydrogenbonds and require more energy to separate them; because the labelingprocedure typically results in short DNA probes, this effect is negligible abovean original length (i.e. prior to labeling) of 500 bp;base composition - because GC base pairs have one more hydrogen bondthan AT base pairs, strands with a high % GC composition are more difficult to

separate than those with a low % GC composition;chemical environment - the presence of monovalent cations (e.g. Na+ ions)stabilizes the duplex, whereas chemical denaturants such as formamide andurea destabilize the duplex by chemically disrupting the hydrogen bonds.


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Southern blot nucleic acid hybridization assays currently in use today use solution hybridization, andinvolve mixing of an aqueous solutions of probe with involved target DNA which has been immobilizedon a solid support, such as a membrane made of nitrocel lulose or nylon. Hybridization of labeled probeto the immobilized target DNA can then be followed by removing the solution containing unbound probeDNA, extensive washing and drying in preparation for detection. This is the basis of the standard. More

recently, however, reverse hybridization assays have become more popular. In these cases, the probepopulation is unlabeled and fixed to the solid support, while the target nucle ic acid is labeled and presentin aqueous solution. Note, therefore, that probe and target are not primarily distinguished by which is thelabeled and which is the unlabeled population. Instead, the important consideration is that the targetDNA should be the complex imperfectly understood population which the probe (whose molecularidentity is known) attempts to query. Depending on the nature and form of the probe and target, a verywide variety of nucleic acid hybridization assays can be devised.

A Dot blot (orSlot blot) is a technique in molecular biology used to detect biomolecules. Biomoleculesto be detected are not first separated by electrophoresis, instead, a mixture containing the molecule tobe detected is applied directly on a membrane as a dot. The technique offers significant savings in timeas gel electrophoresis and the complex blotting procedures for the gel are not required. However, itoffers no information on the size of the target biomolecule as molecules of different sizes when detected,will appear as a single dot. Dot blots therefore can only confirm the presence or absence of abiomolecule or biomolecules. A radioactive sample can be hybridized to it allowing the researcher to

detect variation between samples. The DNA is quantified and equal amounts are aliquoted into tubes inexcess of the number of its targets in the samples, such as 10ug for a plasmid and 1ug for a PCRamplicon. These are denatured (NaOH and 95C) and added to the wells where a vacuum sucks thewater (with NaOH and NH4OAc) from underneath the membrane (nylon or nitrocellulose).


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Radioactive phosphorous atom (32P or33P) in nucleotide strands are detectedby x-ray film or special scanner (e.g. phosphorimager

http://imagers.salk.edu/pimager/pimFAQ.html)

DIG labeled and other antibody detected probes can be visualised usingChemiluminescence detection with CCD camera or Chromogenic methods.


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Fluorescence microscopy and structure of common fluorophores

(A) Structure of fluorophores. The example on top shows fluorescein-dUTP.

The fluorescein group is linked to the 5 carbon atom of the uridine by a

spacer group so that when the modified nucleotide is incorporated intoDNA, the fluorescein group is readily accessible. Below is the structure ofrhodamine from which a variety of fluorophores have been derived.

(B) Fluorescence microscopy. The excitation filter is a color barrier filter whichin this example is selected to let through only blue light. The transmittedblue light is of an appropriate wavelength to be reflected by the dichroic(beam-splitting) mirror onto the labeled sample which then fluoresces andemits light of a longer wavelength, green light in this case. The longerwavelength of the emitted green light means that it passes straight through

the dichroic mirror. The light subsequently passes through a second colorbarrier filter which blocks unwanted fluorescent signals, leaving the desiredgreen fluorescence emission to pass through to the eyepiece of themicroscope. A second beam-splitting device can also permit the light to berecorded in a CCD camera.


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Human chromosome 19 territories, that contain mainly gene-dense and earlyreplicating chromatin, are located toward the nuclear center, whereas chromosome18 territories, which consist mainly of gene-poor and later replicating chromatin, islocated close to the nuclear border.

This is conserved feature of at least seven different primate species irrespective ofthe major karyotypic rearrangements that occurred in different phylogenetic lineages.The evolutionarily conserved positioning of homologous chromosomes orchromosome segments in related species supports evidence for a functionallyrelevant higher-order chromatin arrangement that is correlated with gene-density.

Three-dimensionally reconstructed of chromosomal territories (CTs) in greatapes and white- handed gibbon nuclei.(a) Three-dimensional positioning of two chromosomal loci: HSA18 (red) and HSA19

(green) CTs in a human lymphoblastoid cell nucleus with the partiallyreconstructed nuclear border (outside, blue; inside, silver-gray).

(b, c, d) Three-dimensional positioning of HSA18 and HSA19-homologous CTs innuclei of great apes. In correspondence with the situation in humanlymphoblastoid cell nuclei, the HSA18-homologous CTs were always positionedclose to the nuclear border. The relative locations of the CTs, however, variedfrom a close neighborhood to opposite positions (for example, compare b and c).

(e and f ) Two gibbon nuclei exemplify the variability but still consistently interiorlocation of reshuffled HSA19-homologous chromosome segments in this speciesof lesser apes in contrast to the peripherally located HSA18-homologoussegments.

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Using fluorescence in situhybridization to define a translocation

breakpoint.

(A) Cytogenetically defined translocation t(8;16)(p22;q21). (B) Physical map of

part of the breakpoint region in a normal chromosome 8, showing approximatelocations of seven clones. (C) Results of successive FISH experiments. Thebreakpoint is within the sequence represented in clone D. This result wouldnormally be confirmed using clones from chromosome 16.

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Documents

4023-1 Genome Structure, Organ is at Ions and Methods of Analysis(1)