Chp 7 - Chromosomal Structure and Chromosomal Mutations (From textbook and ppt)

Nomenclature - descriptive characters of abnormalities

+gain of a whole chromosome—47,XY,+21 -loss of a whole chromosome—45,XY,-7 del deletion — 46,XY,del(7)(q22) dup duplication — 46,XY,dup(17)(p12p12) t(n;n) translocations—46,XX,t(2;8)(q21;p13)

Dual-color break apart probes or Tri-FISH probes

0.6 to 1.5 Mb are another approach to decrease background signals as well as to identify translocation events where one chromosome can recombine with multiple potential partners. The probes bind to the intact chromosome flanking the translocation breakpoint. When a translocation occurs, the two probes separate. Sometimes called tri-FISH, break apart probes are not the same as tricolor probes. Normal cells show the combination signal and translocation will separate the probe signals.

Chromosome mutations detected by karyotyping (6 types)

1.Translocations described above. 2. Deletion is a loss of chromosomal material. Large deletions covering millions of base pairs can be detected using karyotyping; smaller microdeletions can't always be seen. 3. Insertion is a gain of chromosomal material. Just like deletions these can cause altered banding patterns and a change in the size of chromosomes. 4. Inversions result from excision, flipping, and reconnection chromosomal material within the same chromosome. Pericentric inversions include the centromere in the inverted region, whereas paracentric inversions involve sequences within one arm of the chromosome. Isochromosome is a metacentric chromosome that results from transverse splitting of the centromere during cell division. Transverse splitting causes two long arms or two short arms to seprate into daughter cells instead of normal chromosomes with one long and one short arm. The arms of an isochromosome are equal in length and genetically identical. 5.A ring chromosome results from deletion of genetic regions from ends of the chromosome and a joining of the ends to form a ring. 6.Derivative chromosome is an abnormal chromosome consisting of translocated or otherwise rearranged parts from two or more unidentified chromosomes joined to a normal chromosome. Fig 7.14 pg 190 lists diff chromosome mutations In addition to translocations, ring and derivative chromosomes may or may not result in loss of chromosomal material. google: A derivative chromosome (der) is a structurally rearranged chromosome generated either by a chromosome rearrangement involving two or more chromosomes or by multiple chromosome aberrations within a single chromosome (e.g. an inversion and a deletion of the same chromosome, or deletions in both arms of a single chromosome)

Interphase chromatin

10 micron fiber is further coiled around histone H1 (or H5 in certain cells) into a thicker and shorter 30nm or 30 micron fiber. The 30nm interphase fibers represent the "resting state" of the DNA. The fibers are locally relaxed into 10nm fibers for DNA metabolism as required during the cell cycle (interphase). When the DNA is relaxed into 10-micron fibers for transcription or replication, the placement of nucleosomes along the double helix can be detected using nucleases ( Mung bean nuclease or DNase I). These enzymes cut the double helix in the part of the double helix that is exposed between the histones.

Order of chromosome compaction

10 nm (active DNA during interphase) ->30 nm (Silent DNA) looped into protein scaffolds to form the 300nm before entry into cell cycle (mitosis) >300nm looped fibers are wound into 700nm solenoid coils->700nm coils are compacted into 1,400 nm fibers->1,400nm

Human Genome consists of ___bp and ___chromosomes

2.9 billion nt base pairs of DNA organized into 23 chromosomes

Normal Male Karyotype

22 pairs of autosomes, one inherited from each parent, and one pair of sex chromosomes, XY. This karyotype is designated 46, XY

Type of chromosome compaction for karyotype

700nm coils are compacted into 1,400 nm fibers that can be seen microscopically in metaphase nuclei.

Variant

A more general term to describe inherited or somatic sequence alterations. "Mutation" term is used for rarer, usually somatic changes, for example, changes only seen in tumor tissue.

Mutation and polymorphism are both

A transmissible (inheritable) change in the DNA sequence. These terms mean different things.

Polymorphism

A variant that is present in at least 1% to 2% of a population. Two or more genetically determined, proportionally represented phenotypes in the same population

29. Comparative genome hybridization detects which type of genetic abnormalities? A. Amplifications relative to reference DNA B. Absolute gene numbers C. Inversions D. Amino acid substitutions

A. Amplifications relative to reference DNA (think it is a comparison between test and reference)

A polymorphism is distinguished from a mutation in what way? A. Its frequency in a given population B. The size of the genetic region involved C. The severity of the effect on phenotype D. Associations with other genetic events

A. Its frequency in a given population. A mutation is defined as any change in a DNA sequence away from normal. This implies there is a normal allele that is prevalent in the population and that the mutation changes this to a rare and abnormal variant. In contrast, a polymorphism is a DNA sequence variation that is common in the population.

Tricolor probe

Addition of CEN probe to dual-color probes comprises a tricolor probe and serves as a control for amplification or loss of one of the chromosomes involved in the translocation. EX: the IGH/MYC/CEP 8 Tri-color probes are a mixture of a 1.5-Mb labeled probe complementary to the immunoglobulin heavy-chain region (IGH) of chromosome 14; a 750 kb distinctly labeled probe complementary to the myc gene on chromosome 8; and a CEN to chromosome 8.

Translocation visualized under microscope shows

An assay has two probes (one green and one orange). There should be two green probes and two orange probes per cell, since there are two copies of each chromosome. A scientist can detect the translocation under a microscope when the two probes are so close together (due to the translocation) that the green probe and the orange probe merge to become one yellow colored probe. This is what the text means when it says that one signal will be lost.

XYY

Aneuploid with polysomy Y (one extra Y chromosome)

Nucleolar organizing region (NOR) staining

Another region-specific staining approach. Chromosomes treated with silver nitrate will stain specifically at the constricted regions, or stalks, on the acrocentric chromosomes.

Which is the proper order for the four phases of the cell cycle? A. G1, S, M, G2 B. G1, S, G2, M C. G2, S, M, G1 D. M, S, G1, G2

B. G1, S, G2, M

30. Before its conversion to a microarray technique, comparative genome hybridization (CGH) was performed by hybridizing labeled DNA to what support? A. Immobilized PCR products B. Normal chromosome spread C. Nitrocellulose membrane D. Agarose gel

B. Normal chromosome spread

Colcemid is used in the procedure of preparing a chromosome spread for karyotype analysis for which purpose? A. To spread the chromosomes out within the cell B. To inhibit microtubule formation C. To induce cells to enter mitosis D. To fix cells to the slide prior to staining

B. To inhibit microtubule formation

Balanced polymorphism

Balanced polymorphism is a situation in which two different versions of a gene are maintained in a population of organisms because individuals carrying both versions are better able to survive than those who have two copies of either version alone

Interphase

Cell contents GROW. During interphase, cellular organelles double in number, the DNA replicates, and protein synthesis occurs. The chromosomes are not visible and the DNA appears as uncoiled chromatin.

C banding (what type of dye and name of staining dye)

Chemical dye. Alkali treatment of chromosomes results in centromere staining. Centromere staining is absent in G-band patterns and may be associated with heterochromatin, the "quiet" or poorly transcribed sequences along the chromosomes that are also present around the centromeres. Euchromatin which is rich in gene activity may not be stained as much as heterochromatin in C banding. The correlation between heterochromatin and staining may also hold for noncentromeric G and Q bands. This association is complicated tho. Cause a variety if procedures and stains can produce identical banding patterns.

G banding (what type of dye and name of dye)

Chemical dye. Chemical dye Giemsa stains in patterns, or G bands, similar to those seen in Q banding. The appearance of G banding differs, depending on the treatment of the chromosomes before staining. Mild treatment (2x standard saline citrate for 60 min at 60 C) yields the region-specific banding pattern comparable to that seen with fluorescent dyes. Used to map structural aberrations and is most commonly used method for analyzing chromosomes. google karyotyping with G banding "During mitosis, the 23 pairs of human chromosomes condense and are visible with a light microscope. A karyotype analysis usually involves blocking cells in mitosis and staining the condensed chromosomes with Giemsa dye. The dye stains regions of chromosomes that are rich in the base pairs Adenine (A) and Thymine (T) producing a dark band. A common misconception is that bands represent single genes, but in fact the thinnest bands contain over a million base pairs and potentially hundreds of genes. For example, the size of one small band is about equal to the entire genetic information for one bacterium."

R banding (what type of dye and name of it)

Chemical dye. Harsher treatment of chromosomes (87C for 10 min then cooling to 70 C) before Giemsa staining will produce a pattern opposite to the G banding pattern called R banding. R bands can also be visualized after staining with acridine orange.

Visualizing Chromosomes

Cytological stains: Feulgen, Wright, and hematoxylin have been used to visualize chromosomes. An advance in was the discovery of fluorescent stains and chemical dyes can react with specific chromosomes regions. This region specific staining forms reproducible patterns where portions of the chromosome accept or reject the stain. For cytogenetics allows id of every chromosome and the direct detection of some chromosomal abnormalities. The region specific staining is cause the reproducible staining patterns occur as a result of defined regional ultrastructures of the mitotic chromsomes. Staining patterns on chromosomes are used for IDing and site location. Heterochromatin stains darkly by G or Q banding and euchromatin stain darkly by R banding. C banding stains centromeres.

26. Which of the following is an advantage of interphase FISH as compared with metaphase FISH? A. The examination of 20 spreads in interphase FISH has increased sensitivity. B. Interphase FISH allows identification of mutations anywhere in the chromosome. C. Interphase FISH allows identification of all chromosomes. D. Results are available faster in interphase FISH procedures.

D. Results are available faster in interphase FISH procedures. (i am guessing cause no culturing?)

Mutation

DNA sequence change that is present in a relatively small proportion of a population. A permanent transmissable change in the genetic material, usually in a single gene

What does 46, XX,del (7)(q13)

Deletion in long arm q of chromosome 7 at region 1, band 3.

Comparative Genome Hybridization (not on study guide)

Detects intrachromosomal amplifications or deletions. Pro: Can identify the location of deletions or amplifications throughout the Genome. The resolution (precise identification of the amplified or deleted region) is not as high as can be achieved with array CGH. Can identify recurrent genomic imbalances not detected by karyotyping. How it works: DNA from test and reference samples is labeled and used as a probe on normal metaphase chromosome spread. The test sample is compared with a normal refernece sample on metaphase spread. Normally test/reference samples are equal. A higher test signal denotes an amplification and a higher reference signal denotes a deletion. (i think this is the important part) Test DNA is isolated and labeled along with reference DNA. Two colorimetrically distinct cyanine dyes, Cy3 and Cy5 are used as fluorescent labels for the test (green) and reference DNA (Red). Cy 3, fluoresces at wavelngth 550 nm, is represented as green and CY 5, fluoresces in the far-red region of the spectrum (650 to 667 nm) is represented as red. Labeling (Attachment of Cy3 or Cy5 dye to the test and reference DNA) is achieved by nick translation or primer extension in which nt covalently attached to the dye molecules are incorporated into the DNA sequences. pg. 195 for dye molecules used. Uses dUTP or dCTP. Poly I is used as has exonuclease actiivty as well as polymerase activity. Separate aliquots of test and reference DNA are labeled with different Cy3 and Cy5 dyes, respectively, before application to normal metaphase spread. Test DNA is partially digested with DNase to produce fragments that will bind efficiently to the denatured DNA in a metaphase chromosome spread. google details "CGH is only able to detect unbalanced chromosomal abnormalities. This is because balanced chromosomal abnormalities such as reciprocal translocations, inversions or ring chromosomes do not affect copy number, which is what is detected by CGH technologies. CGH does, however, allow for the exploration of all 46 human chromosomes in single test and the discovery of deletions and duplications"."The aim of this technique is to quickly and efficiently compare two genomic DNA samples arising from two sources, which are most often closely related, because it is suspected that they contain differences in terms of either gains or losses of either whole chromosomes or subchromosomal regions (a portion of a whole chromosome)." "This is achieved through the use of competitive fluorescence in situ hybridization.""A higher intensity of the test sample colour in a specific region of a chromosome indicates the gain of material of that region in the corresponding source sample, while a higher intensity of the reference sample colour indicates the loss of material in the test sample in that specific region. A neutral colour (yellow when the fluorophore labels are red and green) indicates no difference between the two samples in that location"

Derivative Chromosome vs Translocation

Difference: translocation is the event that takes place. Derivative chromosome is one of the possible results of a translocation event.

Holocentric chromosomes

During cell divison these chromosomes associate with kinetochores along their entire length. (some plants and insects)

Telomere

Each chromosome arm has a unique set of repeat sequences located just before the end of the chromosome which is called the telomere. There is a set of DNA probes specific to telomeres of all human chromosomes. Order goes dna sequence, probe binding site, 100-200 kb telomere associated repeats, telomere 3-20 kb. The binding sites for telomeric probes are unique sequences just next to the telomeric associated repeats and telomeric repeat sequences at end of chromosomes.

Translocations (Two Types)

Exchange of genetic material between chromosomes. There are several types. A)Reciprocal translocation, part of two chromosomes exchange that is, each chromosome breaks and the broken chromosomes reassociate or recombine with one another. Balanced when this type of translocation results in no gain nor loss of chromosomal material.These translocations therefore may occur without phenotypic effects. Balanced translocations in germ cells (cells that give rise to eggs or sperm) can, however, become unbalanced by not assorting properly during meiosis; as a result, they affect the phenotype of offspring. google: "Carriers of reciprocal translocations are phenotypically normal but have problems at meiosis and produce unbalanced gametes. This leads to a reduced litter size or even infertility" B)A robertsonian translocation involves the movement of the long arm of an acrocentric chromosome to the centromere of another acrocentric chromosome. This type of translocation may also become unbalanced during reproduction resulting in a net gain or loss of chromosomal material in the offspring. Results in two p arms attached together and two q arms attached together or an isochromosome. Which is an unbalanced structural abnormality in which the arms of the chromosome are mirror images of each other. The long arms of the two acrocentric chromosomes are joined at the centromere.

Q banding (uses what type of dye and name of dyes)

Fluorescent dye. When chromosomes are stained with fluorescent dyes, quinacrine and quinacrine mustard the resulting fluorescent pattern visualized after staining is Q banding. Each human chromosome could be id by its characteristic banding pattern. Can also distinguish the Y chromosome in humans in interphase nuclei. Because Q banding requires a fluorescent microscope it is not as widely used as other stains that are detectable by light microscopy.

Metaphase FISH

Fluorescent probes bind to metaphase chromosomal regions or to whole chromosomes. Two types: ■Chromosome painting ■Spectral karyotyping Pro: Allows analysis of small regions not visible by regular chromosome banding Multicolor FISH (QMFISH or M-FISH) Combinations of diff specific probes and chromosome paints can be used simultaneously, yielding more info than carrying out separate experiments on multiple specimens. Imaging with 10-20 diff probes or with a combination of region-specific probes and spectral karyotyping differentiates multiple chromosomes by spectral propertires. Reveals structural or numerical abnormalities. Simultaneously, M-FISH identifies specific chromosomal regions based on the presence or abensce of the probe color visualized with specific filters. Analysis by both may show cryptic translocations and insertions as well as the chromosomal origins of marker chromosomes. Special quirks: Whole chromosome paints - are probes that cover the entire chromosome and are valuable for detecting these small or complex rearrangements Spectral karyotyping - is the mixing of combinations of 5 fluors and using special imaging software. Used to distinguish all 23 chromosomes by chromosome-specific colors. Can detect abnormalities that affect multiple chromosomes, as is sometimes found in cancer cells or immortalized cell lines. Telomeric and centromeric probes are also applied to metaphase chromosomes to detect aneuploidy and other genomic mutations. Preparation: Culture cells for 72 hours. About 45 min before harvesting, colcemid is added to the cultures to arrest dividing cells in metaphase. The cells are then suspended in a hypotonic medium (0.075 M KCL) and fixed with methanol/acetic acid (3:1). The fixed-cell suspension is applied to an inclined slide and allowed to dry. A second treatment with 70% acetic acid may improve the chromosome spreading and decrease background noise. Under a phase contrast microscope the chromosomes should appear well separated with sharp borders. Cytoplasm should not be visible. Once the slide is dried, hybridization proceeds as it did in interphase-FISH. Con: Condensed chromosome spreads, especially those from cultured metaphases, may be affected by temp and humidity. Uses of Fluorescent in situ Hybridization (FISH) ■Identification and characterization of numerical and structural chromosome abnormalities ■Detection of microscopically invisible deletions ■Detection of subtelomeric aberrations ■Prenatal diagnosis of the common aneuploidies (interphase FISH)

Quantitative FISH (Q-FISH)

Fluorescent signals are digitized and quantified. The relative intensity of signals in multiple loci will demonstrate copy-number variations. Analysis of repeated sequences by assessing the relative intensity of probe signals. Microscopic images are digitized on a charge-coupled device (CCD). The signals are then measured by imaging software, quantifying FIShH signals from each digital image. The relative intensity is compared between signals. The telomere Q-FISH technique has been applied to studies on telomere length.

Position Effect

Genes with identical DNA sequence will behave differently depending on the chromosomal location or the surrounding nt sequence of the gene. A gene inserted or moved into a diff chromosomal location may be expressed (transcribed and translated) differently than it was in its original position. Also - Different sequences can have the same functional effect, such as centromeres, which are not defined by specific DNA sequences. I guess can affect chromosomes in a similar way as centromere are involved in the separation of chromsomes during cell divison. Could cause gene to be in diff location or next to diff nt sequences i suppose.

Types of Mutations

Genomic: abnormal chromosome number (monosomy, polysomy, aneuploidy). Polysomy -organism has at least one more chromosome than normal, i.e., there may be three or more copies of the chromosome rather than the expected two copies.Aneuploidy is the presence of an abnormal number of chromosomes in a cell, for example a human cell having 45 or 47 chromosomes instead of the usual 46. Chromosomal: abnormal chromosome structure Gene: DNA sequence changes in specific genes

Humans are diploid or haploid?

Humans are diploids who inherit a haploid set of genes (23 chromosomes) from each parent, so humans have 2 copies of every gene (except for some on the X or Y chromosome). Each chromsome is a double helix of DNA ranging from 246 million nt bp in length in chromosome 1 to 48 million nt bp in chromosome 21. Genetic information is carried on the chromosomes in the form of the order or sequence of nucleotides in the DNA helix. A phenotype is a trait or group of traits resulting from transcription and translation of these genes. The genotype is the DNA nucleotide sequence responsible for a phenotype.

Centromeric Probes (CEN) Probes

Hybridize to highly repetitive alpha satellite sequences surrounding centromeres. These probes detect aneusomy of any chromosome. Combinations of centromeric probes and region-specific probes are often used to confirm deletions or amplifications in specific chromosomes. Used in both Interphase and Metaphase FISH.

FISH vs Karyotyping

Inability to identify chromosomal changes other than those at the specific binding region of the probes. Karyotyping is a more generic method which can detect any chromosomal change that causes changes in chromosomal size, number, or banding pattern within the sensitivity limits of the procedure.

Two Types of Fluorescence In Situ Hybridization FISH

Interphase and Metaphase

DNA replication and RNA transcription both occur during

Interphase. Prior to mitosis. DNA replication and protein synthesis, which includes transcription and translation, occurs throughout interphase

Metaphase Chromatin is made up of

Is one-third DNA, one third histone, one third non-histone proteins. Nonhistone protein complexes, termed condensin I and condensin II, maintain mitotic chromosome structure. Histones wrapped in DNA forms the 10-nm DNA prior to metaphase this is active DNA for replication and transcription. Further compaction results in the closed 30nm fibers found in transcriptionally silent DNA.

Detection of Genome and Chromosomal Mutations (5 answers)

Karyotyping, Fluorescence In Situ Hybridization, Interphase FISH, Metaphase FISH, Comparative Genome Hybridization (CGH)

Identification of chromosomal location by G-band patterns

Locations are designated by the following example chromosome number 17, the arm q, the region 1, the band 1, and the sub-band 2. (17q11.2)

Making of Karyotypes

Manual assembly of karyotypes from light microscopic images has been replaced by software systems that electronically arrange the chromosomes from the image of the chromosome spread. This type of image is more difficult to apply to fluorescent chromosome analysis which would require a motorized scanning stage, automated area selection on slide, and signal evaluation.

Maintenance of heterochromatin throughout interphase

May require condensin proteins or condensin like protein complexes (nonhistone proteins). Family of proteins called SMC proteins control chromosme condensation in eukaryotes and chromosome segrgation in prokaryotes. XCAP-C and CAP-E first isolated from frog eggs are integral parts of the condensin complex, a protein scaffold structure that can be isolated from both mitotic and interphase cells. In the presence of topoisomerase this complex can wrap DNA around itself in a reaction driven by ATP. SMC Family proteins play a role in repair of chromosmal breaks.

Chromosome naming (4 names)

Metacentric, submetacentric, acrocentric, or telocentric depending on the placement. The placement of the centromere divides the chromosome into arms. Metacentric chromosome arms are equal in length. Submetacentric one arm is longer than the other. One arm is extremely small or missing in acrocentric or telocentric chromosomes.

FISH What is it?

Method used to detect protein and RNA as well as DNA structures in place in the cell, or in situ. TLDR: Hybridization of complementary gene- or region specific fluorescent probes to chromosomes. Will see two signals one for each chromosome inside cell nucleus. Two red means two copies of that gene. More rapid assay with high resolution and flexibility than karyotyping Targets specific sequences of chromosomes with fluorescent probes. Limited to regions complementary to the FISH probes Probes are designed to hybridize to critical areas that are amplified, deleted, translocated or otherwise rearranged in disease states Kayotyping is performed using light microscope FISH requires fluorescence microscope that will excite fluorescent emission for the probes and special filters for detection of fluors emitting at different wavelengths.

Chromosome Morphology

Mitotic chromosomes have been distinguished by their relative size and centromere placement. For example for classification: Large (Size) metacentric (centromere placement) for chromosomes 1 and 2. It seems chromosome number is inverse to the size. So lower number chromosomes are larger.

Higher Order Repeat HOR

Monomer = AAAAAA, TTTTTTT, GGGGG or CCCCCC Higher order repeat= AAAAAATTTTTTT Higher order repeat array = AAAAAATTTTTTTAAAAAATTTTTTT

3 Categories of mutations

Mutations can affect a single NT or millions of NT, even whole chromosomes and thus can be classified as gene, chromosome, and genome mutations. Gene mutations affect single genes and typically are small changes in the DNA sequence. Chromosome mutations affect the structures of large chromosomal regions (hundreds of thousands to millions of base pairs) either within the same chromosome or to another chromosome. Genome mutations are changes in the number of chromosomes. A cell or cell population with a normal complement of chomrosomes is euploid. Genome mutations result in cells that are aneuploid.

Benign polymorphism

No selective advantage. Examples: ABO blood groups and major histocompatibility complex and polymorphism used for human identification and paternity testing.

Number of chromosomes per nucleus

Normal number is 46

Indicator of apoptosis

Normally in 30 nm interphase chromatin fiber, the internucleosomal DNA is wound into a solenoid coil. Loss of this level of organization is indicator of apoptosis. The 30 nm fibers are uncoiled and the exposed linked DNA between the nucleosomes becomes susceptible to digestion by intracellular nucleases. The DNA wrapped into the nucleosomes remains intact so that DNA isolated from apoptotic cells contains "ladders" or discrete multiples of approximately 180 bp. These ladders were used in earlier molecular methods to detect/confirm cell death by apoptosis.

Other types of staining

Prior to Giemsa, Trypsin or other proteolytic extraction or denaturation proteins were used.

Interphase FISH

Pro:Unlike Karyotyping does not require culturing of cells. Commonly used to study prenatal samples, tumors, and hematological malignancies They are brought into metaphase in culture. Valuable for analysis of cells that do not divide well as don't need to culture thus for fixed cells. Hundreds of cells are analyzed microscopically so the sensitivity of detection is higher than that of metaphase procedures which commonly examine 20 spreads. How it works: Fixed cells are permeabilized and exposed to a probe. Probe is 60 - 200 kb fragment of DNA attached covalently to a fluorescent molecule. Bound probe is visualized under a fluorescent microscope as a point of fluorescent light in the nucleus of the cell. Probes are complementary to a particular chromosome or chromosomal locus so that the image under the microscope will correlate with the state of that chromosome or locus. A probe to chromosome 22 would yield two signals per nucleus as two copies of chromosome 22. Normal nucleus will have two of each of the probe signals. Multiple probes spanning large regions are used to detect regional deletions. Translocations or other rearrangements are detected using probes of different "colors" (or signals) complementary to regions on each chromosome taking part in the translocation. A translocated chromosome will combine two of the probe signals, resulting in a loss of one of each signal in the nucleus. More detail on cell prep: Want to prepare cells and maintain cell morphology. Fresh interphase cells are incubated overnight (aging) after deposition on slides. Then cells are treated with protease to minimize interference from cytoplasmic proteins and fixed with 1% formaldehyde to stablize the nuclear morphology. Before DNA denaturation, the cells are dehydrated in graded concentrations of ethanol. Paraffin-embedded fixed tissues are dewaxed in xylene before protease and formaldehyde treatment. Fluorescent probes (DNA with covalently attached fluorescent dyes) can come from vendors which may also supply compatible hyb reagents and controls. However, the probe performance should be observed on control tissue before use on patient samples. The quality of the probe must be checked and its performance validated before use. Use controls with each run of samples as a given probe can differ in their signal characteristic and intensities based on the diff types of tissues. Just like with blots, both probe and target must be denatured prior to hybridization. The amount of time taken to hybridize and use Cot-1 DNA (placental DNA enriched for repetitive sequences to reduce nonspecific binding) or facilitators such as dextran sulfate (to increase the effective probe concentration) depends ont he sequence complexity of the probe. 1-10 ug of probe may be used in a hyb volume of 3 - 10ul. The hybridization of the probe on target cells is performed at 37-42 C in a humidified chamber. The slides are cover-slipped and sealed to optimize the hybridization conditions. Following hybridization and rinsing off of the unbound probe, the sample is observed microscopically. Cons: False signals that result from two two chromosomes landing close to one another in the nucleus. Bound probes give signal similar to translocation. Can distinguish by size of the fluorescent image or vertical focusing with the microscope. Accounting for false-positive signals as background noise limits the sensitivity of this assay. Adequate numbers of cells must be visible, but crowded cells where the nuclei and signals overlap do not yield accurate results. Also, diff tissue types have diff image qualities and characteristics that must also be taken into account with FISH image. With FISH analysis can also have photobleaching (Fading) or loss of probe signal emission (10^5 photons/second) due to photochemical destruction of the fluorophore molecules. Thus FISH slides should not be subjected to prolonged light exposure.

Robertsonian translocation

Robertsonian translocation is a type of translocation caused by breaks at or near the centromeres of two acrocentric chromosomes. The reciprocal exchange of parts gives rise to one large metacentric chromosome and one extremely small chromosome that may be lost from the organism with little effect because it contains few genes. The resulting karyotype in humans leaves only 45 chromosomes, since two chromosomes have fused together.[7] This has no direct effect on the phenotype, since the only genes on the short arms of acrocentrics are common to all of them and are present in variable copy number (nucleolar organiser genes). A balanced translocation isn't between two acrocentric chromosomes. It results in two balanced chromosomes. No material lost.

Dual-Fusion Probes

Sensitivity for translocation is increased through the use of dual-color probes, or dual-fusion probes. They are mixtures of two single probes, each labeled with a diff fluorescent dye. They bind to regions spanning the breakpoint of translocations. A translocation will be observed as a signal from both the translocation junction and the reciprocal of the translocation junction for example t(9;22) and t(22;9). A normal nucleus has two signals for each probe. A translocation involving the two chromosomes combines the two probe colors. Dual-fusion probes confirm the presence of the translocation by also giving signal from the reciprocal breakpoint.

Histones role

Structural role and control access to and expression of DNA. Modification through acetylation, methylation, phosphorylation, or ubiquitination, alters DNA access and plays a role in other cellular functions such as recombination, replication, and gene expression.

Compaction of DNA

The winding of DNA onto histones (most abundant protein in the cell) is the first step in compaction. 160-180 bp of DNA are wrapped around a set of 8 histone proteins (Two of each of H2a, H2b, H3, and H4 to form a nucleosome.

Down Syndrome

Trisomy 21 (47, XX or XY, +21)

Telomeric Probes

Useful for the detection of chromosome structural abnormalities, such as cryptic translocations or sub-telomeric deletions that are not easily visualized by standard karyotyping. Used in both Interphase and Metaphase FISH.

Nucleosomes

Visible by electron microscopy as 100-A beadlike structures that ares separated by short (70-90bp) strands of a free double helix or linker DNA.

DAPI (4',6-diamidino-2-phenylindole)

Way to detect mycoplasmal contamination in cell cultures. DAPI binds to the surface grooves of ds DNA and fluoresces blue under UV light (352-nm wavelngth). Used to visualize chromosomes as well as whole nuclei.

Klinefelter syndrome

XXY

Eukaryotic chromosome is

a double helix of DNA. A cell nucleus contains 4 cm of double helix, which must be compacted both to fit into the nucleus and to accurately segregate in mitosis. An extended DNA double helix undergoes an 8,000-fold compaction to make a metaphase chromosome.

Mitosis (metaphase is part of this)

a type of cell division that results in two daughter cells each having the same number and kind of chromosomes as the parent nucleus, typical of ordinary tissue growth

Human Chromosomes are

acrocentric or submetacentric and so have long and short arms. Confusing as table has metacentric as well. The long arm of a chriomosome is designated q and the short arm is designated p. Acrocentric chromosomes have a ratio of long arm length: short arm length from 3:1 to 10:1. Chromosomes 13-15,21,22 are considered acrocentric but may be classified as subtelocentric.

Polymorphisms

are casually considered mutations that do not severely affect phenotype; that is generally true because any negative effect on survival and reproduction limits the persistence of a genotype. Exceptions exist like sickle cell anemia where balance of positive and negatives help it to be maintained in population is example of mutation but really is a balanced polymorphism. Internet: Genetic polymorphism refers to the occurrence of two or more genetically determined phenotypes in a certain population. A gene is said to be polymorphic if more than one allele occupies that gene's locus within a population (see two pictures on desktop explaining alleles on a chromosome). Genes that control hair color are polymorphic.

Chromosome behavior is dependent on

chromosomal structure as well as DNA sequence.

Somatic mutation

genetic alteration acquired by a cell that can be passed to the progeny of the mutated cell in the course of cell division. Somatic mutations differ from germ line mutations, which are inherited genetic alterations that occur in the germ cells (i.e., sperm and eggs). Somatic mutations are frequently caused by environmental factors, such as exposure to ultraviolet radiation or to certain chemicals.

Satellite DNA

google "Satellite DNA consists of arrays of tandemly repeating sequences that are widely present (>30%) in the genome. Satellite DNA is mainly present in heterochromatin or the tightly packed regions of chromosomes in centromeres, telomeres, and sometimes even in the euchromatin region (active region of the genome)"

Correlation of staining with heterochromatin

is contradicted by observation of X chromosome. Although one X chromosome is inactive and replicates later than the active X in females, both chromosomes stain with equal pattern and intensity. Staining difference must be due to other factors.

Aneuploidy

is mostly observed as increased numbers of chromosomes because the loss of whole chromosomes is generally not compatible with survival. Aneuploidy in diploid organisms can result when there are more than two copies of a single chromosome or when there are multiple copies of one or more chromosomes. Ex: Down syndrome is an example of a disease resulting from aneuploidly where there are three copies, or trisomy, of chromosome 21.

Both mutations and polymorphisms

may or may not produce phenotypic differences.

Some changes in chromosome structure/ number can be seen

microscopically. Mutations at the nt-sequence level are detected using biochemical or molecular methods. Direct visualization of genome and chromosomal mutations under the microscope and molecular methods to detect single base changes.

High-resolution banding

number of visualized bands can be increased from about 300 to 500 per chromosome by staining chromosomes before they reach maximal metaphase condensation

Genotypic analysis

performed to confirm or predict phenotype

Centromere

site of attachment of the chromosome to the spindle apparatus. The connection is made between microtubules of the spindle and a protein complex kinetochore that assembles at the centromere sequences. At the nt level, the centromere is composed of a set of highly repetitive alpha satellite sequences. These repetitve sequences interfere with chromosome compaction so that microscopically the centromere appears as a constriction in the metaphase chromosome. The centromere consists of tandem repeats of 171 bp sequences flanking sets of single repeat units or monomers repeated in groups in a higher order repeat array. The kinetochore is a protein structure that connects the centromere chromatin to the spindle apparatus. google "Alpha satellite DNA (AS) is the most abundant tandem repeat DNA found in the centromeres of simian primates. The AS of humans contains sequences organized into higher-order repeat (HOR) structures, which are tandem arrays of larger repeat units consisting of multiple basic repeat units."

Chromosome banding facilitates

the detection of deletions, insertions, inversions, and other abnormalities and the identification of distinct chromosomal locations. For this purpose G-banding pattern has been ordered into regions comprising bands and subbands.

FISH Probes

■Chromosome-specific centromere probes (CEP® ) -Hybridize to centromere region -Detect aneuploidy in interphase and metaphase ■Chromosome painting probes (WCP) -Hybridize to whole chromosomes or regions -Characterize chromosomal structural changes in metaphase cells -WCP detects translocation during Metaphase ■Unique DNA sequence probes (LSI® ) Locus Specific Identifier Fusion and Break apart probes! -Hybridize to unique DNA sequences -Detect gene rearrangements, deletions, and amplifications -Greater or fewer than two signals per nucleus is considered abnormal. So if see 3 of same color is triploidy or insertion and if see 1 monosomy is monosomy or deletion ■Telomere-specific probes (TEL) -Hybridize to subtelomeric regions -Detect subtelomeric deletions and rearrangements

Chromosome Morphology Changes During the Cell Division Cycle

■DNA double helix: 2 nm diameter Interphase (G1, S, G2) ■Chromatin "beads on a string": 11 nm ■Chromatin in nucleosomes: 30 nm Metaphase (mitosis) ■Extended metaphase chromosomes: 300 nm ■Condensed metaphase chromosomes: 700 nm

Karyotyping

■Karyotyping detects changes in chromosome number and large structural changes. Genome mutations, or aneuploidy can be detected by indirect methods such as flow cytometry and more directly by karyotyping. A karyotype is the direct observation of metaphase chromosome structure by arranging metaphase chromosomes according to size. Requires collection living cells and growing them in culture in the laboratory for 48 to 72 hours. Cell division is stimulated by addition of a mitogen, usually phytohemagglutinin. Dividing cells are then arrested in metaphase with Colcemid, an inhibitor of microtubule (mitotic spindle) formation. The chromosomes in dividing cells that arrest in metaphase will yield a chromosome spread when the cell nuclei are disrupted with hypotonic buffer. The 23 pairs of chromosomes can then be assembled into an organized display, or karyotype, according to their size and centromere placement. Aneuploidy may be observed affecting several chromosomes ( numbers 5,12,6,9,10) or a single chromosome (y chromosome) Can also detect chromosomal mutations such as translocations etc. See additional flashcards.

Karyotyping vs. FISH

■Karyotyping detects changes in chromosome number and large structural changes.■Structural changes include translocation, duplication, and deletion of chromosomal regions.■More subtle chromosomal changes can be detected by metaphase or interphase FISH.

Visualizing Metaphase Chromosomes

■Patient cells are incubated and divide in tissue culture. ■Phytohemagglutinin (PHA): stimulates cell division ■Colcemid: arrests cells in metaphase ■3:1 methanol:acetic acid: fixes metaphase chromosomes for staining■Chromosomes are analyzed by Giemsa staining during Metaphase (black banding patterns) and karyotyping (probes to a spread)