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ASCP MB Molecular Diagnostics Chapter 8
Chromosome Structure and Mutations
34
Biology
Professional
02/24/2016

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Term
Haploid Genome
Definition
23 Chromosomes
2.9 billion bp

Longest Chromosome - Chromosome 1, 246 million bp

Shortest Chromosome - 47 million bp
Term
Mutation vs. Polymorphism
Definition
Mutation - Change in DNA sequence found in a small proportion of the population. May be meant specifically as somatic changes, such as those in tumors.
Variant - Similar to mutation, but may be a specifically inherited alteration.
Polymorphism - DNA sequence alteration found in 1-2% of the population. May be benign, harmful, or balanced (e.g., sickle cell anemia).
These may or may not produce phenotypic differences.
Term
Levels of Mutation
Definition
Gene mutation - Found in a single gene, usually a small change.
Chromosome Mutation - Affects whole chromosome structure, with movements of large sections within or among chromosomes.
Genome mutation - Change in number of chromosomes.
Term
DNA Compaction
Definition
DNAdouble helices are 2 nm I width. These are wrapped around nucleosomes in 11 nm wide "bells on a string" chromatin, which is wrapped around histone H1 into a 30 nm packed fiber. This is what chromatin at interphase is like. Chromatin is packed onto protein scaffolds to make 300 nm fibers before the M phase and then further wound to make 700 nm solenoid coils.
Metaphase nuclei contain solenoid coils compacted into 1400 nm fibers.
Term
Apoptotic DNA
Definition
If the 30 nm interphase chromatin fibers associate with H1 are uncoiled at the wrong time, it can expose the linker DNA to nucleases for digestion. This is an indicator of apoptosis. The NA wound into nucleosomes remains intact as "ladders" and can be resolved on gels.
Term
Centromeres
Definition
Where spindle apparatus attaches via a protein complex (kinetochore). Highly repetitive alpha sequences of 171 bp flank single repeat units to interfere with chromatin packing and create a constriction at the centromere.
Metacenter centromeres are in the middle of the chromosome.
Submetacentric ones are closer to one end.
Acrocentric ones are very close to one end.
Teleocentric chromosomes lack one arm.
Term
Q Banding
Definition
Fluorescent dyes quinacrine and quinacrine mustard stain heterochromatin darkly. Stains the Y chromosome intensely, can differentiate it even in interphase nuclei. This method is hard to use as it requires a fluorescent microscope. Once of the first banding techniques used, showing that chromosomes have unique banding patterns.
Term
G Banding
Definition
Giemsa dye stains heterochromatin darkly and gives similar patterns to Q banding. Mild treatment of chromosomes in2x saline citrate for 60 min at 60 C before staining yields region-specific banding. Use of denaturing agents like trypsin before staining shows aberrations clearly and is commonly done.
High-resolution banding - DNase I is used before staining with Feulgen to increase visualized bands from 300-500 per chromosome before chromomeres reach maximum compaction.
Term
R Banding
Definition
Treating chromosomes at 87 C for 10 minutes before cooling to 70 C before Giemsa staining yields R bands that are opposite to G bands. Stains euchromatin darkly. Treating with acridine orange visualizes bands.
Term
C Banding
Definition
Treatment of chromosomes with alkali solution to stain centromeres and some heterochromatin, but not euchromatin.
Term
Nucleolar Organizing Region Staining
Definition
NOR. Silver nitrate stains chromosomes specifically at constricted regions of acrocentric chromosomes.
Term
DAPI
Definition
4'6-diamidino-2-phenylindole. Can detect mycoplasmal contamination in cell cultures. DAP fluoresces blue under UV light (353 nm) and binds to dsDNA surface grooves. Can visualize chromosomes or whole nuclei.
Term
Karyotpe
Definition
Direct set of chromosomes in a cell, arranged by size.
Living cells are collected and grown in culture 48-72 hours. A mitogen (usually phytohemaglutinin) is added to stimulate cell division followed by colcimid to arrest cells in metaphase by inhibiting microtubule formation. Cell nuclei are disrupted with hypothec saline and the 23 pairs of chromosomes can be arranged by size. Aneuploidies and insertions/deletions/translocations of size can be detected.
Term
Translocations
Definition
Exchange of genetic material between chromosomes.
Reciprocal translocation - Two chromosomes break and exchange broken pieces. If no genetic material is gained or lost, there may be no phenotypic effect because it is balanced. If balanced translocations in germ cells don't assort correctly during meiosis, the translocation can become unbalanced and have a phenotypic effect.
Robertsonian Translocation - Most of one chromosome moves to the centromere of another. These can become unbalanced during meiosis and result in chromosome loss or gain.
Term
Deletions and Insertions
Definition
Deletion - loss of genetic material.

Insertion - gain of genetic material. A region within the chromosome may be replicated or fragments from other chromosomes may insert themselves.
Term
Inversions
Definition
Chromosomal material is excised, flipped, and reconnected with the same chromosome.
Pericentric inversions invert the centromere.
Paracentric inversions only involve a sequence on one arm or the other.
Term
Isochromosome
Definition
The centromere splits transversely and producs a metacentric chromosome during cell division. This may result in two long arms or two short arms that split into daughter cells, instead of the usual long and short combination. Isochromosome arms are identical in length and sequence.
Term
Ring Chromosome
Definition
Material from both ends of a chromosome are deleted and the two ends are joined, forming a ring.
Term
Derivative Chromosome
Definition
Translocated parts of chromosomes attach to the end of a normal chromosome, forming an abnormal chromosome.
Term
FISH
Definition
Fluorescence in situ hybridization. Can detect proteins, DNA, or RNA in place in the cell.
Term
Interphase FISH
Definition
Doesn't require cell culturing. Used for prenatal samples, tumors, hematological malignancies, all of which are hard to bring to metaphase in culture.
In cytogenetic analysis, probes of 60-200 kb attached to fluorescent molecules are added to fixed cells where they bind to complimentary DNA sequences. A fluorescent microscope will visualize these sequences as a point in the nucleus. Probes are specific for chromosomes, so deletions or duplications are observed.
Term
FISH Detection of Translocation
Definition
Interphase FISH> Probes with different signals complimentary to regions involved in the translocation are used. Normal chromosomes give two of each signal, translocated chromosomes combine two signals, leaving only 3 signals in the cell. If two chromosomes land close together this can complicate results. Size of fluorescent signal can differentiate true from false positives, but it's hard. Sensitivity is lowered by background noise.
Term
FISH Dual Fusion Probes
Definition
Term
FISH Break-Apart Probes
Definition
Interphase FISH. Probes of two different colors bind one each to the two sites flanking the translocation breakpoint. If the chromosome is intact, a combined signal is produced. If there is a translocation, the two colors separate.
This decreases background signal and allows for detection of translocations with multiple chromosome partners.
Term
Centromeric Probes
Definition
Interphase FISH. Hybridize to highly repetitive alpha sequences surrounding centromeres, so aneuploidy of any chromosome can be detected. These can be used in addition to region-specific probes to confirm deletions or amplifications. CEN Probes along with dual color probes make tricolor probes and can be used to control for loss or gain of chromosomes involved in a translocation.
Term
Telomeric Probes
Definition
Interphase FISH. Bind to unique set of DNA sequences before the telomere. Have been designed for all the human chromosomes. Can detect structural abnormalities (cryptic translocations, small deletions).
Term
Benefits of Interphase FISH
Definition
-No cells need to be cultured, so cells that don't culture well or can't divide (fixed tissue) can be analyzed.
- Can examine hundreds of cells in a spread, leading to better sensitivity than metaphase FISH.

-Can't identify chromosomal changes on chromosomes not bound to by the probe, whereas karyotypes will show abnormalities on any chromosome.
Term
Preparation of Interphase FISH Samples
Definition
Cells must be permeabilized and cell morphology must be maintained.
- Fresh interphase cells incubated overnight on slides are best. Fixed cells will need to be dewaxed in xylene.
- Cells are treated with protease.
- 1% formaldehyde fixes the cells and stabilizes nuclear morphology.
Term
FISH Probe Hybridization
Definition
- Probe and target must be denatured first.
- Facilitators like dextran sulfate (increases effective probe concentration) can be used.
- Time to hybridize depends on probe complexity.
- 10 ng -1 ug of probe needs hybridization volume of 3-10 uL at temperature of 37-42 C.
Term
Complications of FISH Analysis
Definition
- Crowded cells where nuclei and cells overlap yield poor results.
- Different tissue types have different qualities and characteristics.
- Photochemical destruction of fluorophor molecules results in photobleaching or loss of probe signal emission (10^5 photons/sec). Can be avoided by limiting light exposure.
Term
Metaphase FISH
Definition
Improves metaphase analysis via probes designed for metaphase chromosomal regions or whole chromosomes.
Whole chromosome paints can detect small or complex rearrangements.
Spectral karyotyping uses five fluors and software to recognize all 23 chromosomes and detect abnormalities affecting multiple chromosomes.
Telomeric/centromeric probes are used to detect deletions/amplifications.
Term
Metaphase FISH Sample Preparation
Definition
- Cells are cultured for 72 hours..
- Colcemid added 45 minutes before harvesting to arrest cells in metaphase.
- Cells are suspended in a hypotonic medium (0.075 MKCl).
- Cells fixed with methanol/acetic acid (3:1).
- Second 70% acetic acid treatment reduces noise.
- Temperature and humidity can affect condensed chromosome spread.
- Chromosomes should separate well with clear borders. Cytoplasm shouldn't be visible.
Term
Comparative Genome Hybridization
Definition
CGH. DNA from test and reference samples is isolated, labeled, and used as a probe on normal metaphase chromosome spreads. Can identify deletions/amplifications through the whole genome. Resolution is not good.
Term
CGH Sample Preparation
Definition
Test and reference DNA are isolated and labeled with Cy3 and Cy5, distinct cyanine dyes. Cy3 fluoresces at 500 nm "green", Cy5 at 650-667 nm, "red". Nick translation or primer extension incorporates nucleotides attached to dye molecules. Separate aliquots of test and reference DNA are respectively labeled with different Cy3, Cy5 dyes. DNase partially digests test DNA to produce fragments to bind to metaphase chromosome spread.
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