The human genome consists of 2.

9
billion nucleotide base pairs of DNA
organized into 23 chromosomes.

Alterations of the DNA sequence

may affect not only the phenotype
of an individual but the progeny of
that individual as well
A transmissible (inheritable) change
in the DNA sequence is a mutation
or polymorphism.
DNA mutations can affect a single nucleotide or millions of nucleotides, even whole
chromosomes, and thus can be classified into three categories: gene, chromosome, and
genome mutations.

Euploid - A cell or cell population with a normal complement of chromosomes

Aneuploid - Aneuploidy is mostly observed as increased numbers of chromosomes because the loss of whole
chromosomes is generally not compatible with survival.
§ An extended DNA double helix
undergoes an 8,000-fold compaction to
make a metaphase chromosome
§ Chromosome topology (state of compaction
of the DNA double helix) affects gene
activity.
§ Chromosome Morphology
§ Conventional cytological stains:
Feulgen
This allows unequivocal identification of every
Wright chromosome and the direct detection of some
Hematoxylin chromosomal abnormalities.

Fluorescent dyes
- quinacrine
Q-banding - Required fluorescent microscope, not widely used.
- quinacrine mustard
v human chromosome could be identified by its characteristic banding
pattern.
Chemical dye - Giemsa
- G bands, similar to those seen in Q banding.
R bands can also be visualized after staining with acridine
orange.
Alkali treatment of chromosomes results in centromere
staining, or C banding.

What are bands?

- The alternating light and dark stained demarcations
which appear along the length of each chromosomes.
- The banding pattern are specific for each
chromosome pair.
§ Staining – dyes bind to the DNA or protein of a chromosome and allow visualization by light microscopy.

§ Giemsa – routine stain

§ Special staining
• Q – banding / quinacrine flourescence staining – routine karyotyping but replaced with G-banding
§ which allow permanent stain preparation.
§ - Rapid identification of Y chromosome
• C- banding – to evaluate constitutive heterochromatin or to determine if the chromosomes has two
§ centromeres.
§ - Centromeres appear as single dark spot
• R- banding – the telomeres should stain as dark and their absence as the result of a deletion is more
obvious.
§ A karyotype is the complete set of chromosomes in a cell.

§ Karyotyping

v is the direct observation of metaphase chromosome structure by arranging metaphase chromosomes according to size.

v can also be used to detect chromosomal mutations such as translocations, which are the exchange of genetic material
between chromosomes.
§ Routine studies – heparinized blood.
§ Hematologic disorders – bone marrow samples
§ Fibroblast cultures from skin biopsies
§ Not routinely done due its invasive in nature
- liver, kidney, lung, and muscles
§ Amniotic fluid – prenatal analysis
- done between 16 – 18 weeks of gestation (20-30ml)
Chronic villus sampling – tissues from the developing placenta. 10 - 14 weeks of
gestation
Cordoncentesis or percutaneous umbilical cord sampling >20 weeks of gestation
• All samples must be collected in a sterile manner.
• Must be transported to the laboratory as soon as possible.
• Blood, bone marrow, amniotic fluid and chronic villi – room temperature
• Solid tissues – wet ice

§ Cell culture technique

§ Bone marrow and blood – aliquoted directly in culture medium

§ Amniotic fluid cells, CV and solid tissues - grown as monolayer in situ.
§ Amniotic fluid – centrifuge
§ 5-10 days – typical culture time. harvested
§
• Swelled hypotonically and are then fixed
v Automated robotic harvester has been developed
§ Types of translocation
1. Reciprocal - each chromosome breaks, and the broken chromosomes reassociate or
recombine with one another.
- When this type of translocation results in no gain nor loss of chromosomal material =
BALANCED
2. Robertsonian translocation - involves the movement of the long arm of an acrocentric
chromosome to the centromere of another acrocentric chromosome = UNBALANCED
Deletion - is a loss of chromosomal material. Large deletions covering millions of base pairs
can be detected using karyotyping
- Microdeletion not usually detected.
Insertion - is a gain of chromosomal material.
Inversions - result from excision, flipping, and reconnecting chromo- somal material within
the same chromosome.
Pericen- tric inversions include the centromere in the inverted region, whereas paracentric
inversions involve sequences within one arm of the chromosome. An iso- chromosome is a
metacentric chromosome that results from transverse splitting of the centromere during cell
division.
§ ring
chromosome results from deletion of genetic regions from ends of the
chromosome and a joining of the ends to form a ring.
§ derivative chromosome is an abnormal chromosome consisting of
translocated or otherwise rearranged parts from two or more unidentified
chromosomes joined to a normal chromosome.
Results of karyotyping analyses are expressed as the number of chromosomes per nucleus
(normal is 46), the type of sex chromosomes (normal is XX or XY), followed by any genetic
abnormalities observed. A normal karyotype is 46,XX in a female or 46,XY in a male.
§ is a method widely used to detect protein and RNA as well as DNA structures in place in the cell, or in situ.

§ more rapid assay with higher resolution and flexibility than karyotyping.

§ it is limited to the regions complementary to the FISH probes.

§ FISH analysis requires a fluorescence microscope that will excite fluorescent emission for the probes and special filters for
detection of fluors emitting at different wavelengths.
§ requires expertise
§ are used commonly to study prenatal samples, tumors, and hematological malignancies

§ fixed cells are permeabilized and exposed to a probe.

§ The probe is a 60- to 200-kb fragment of DNA attached covalently to a fluorescent molecule.

§ Probes are designed to be 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.
v dual-color probes, or dual-fusion probes.
§ Centromeric probes (CEN probes) are designed to hybridize to highly repetitive alpha satellite sequences surrounding
centromeres. These probes detect aneusomy of any chromosome.
§ Telomeric probes are useful for the detection of chromosome structural abnormalities, such as cryptic translocations or sub-
telomeric deletions that are not easily visualized by standard karyotyping.
incubated overnight (aging) after deposition on slides.

treated with protease

(to minimize interference from cytoplasmic proteins)
fixed with 1% formaldehyde to stabilize the nuclear morphology.

dehydrated in graded concentrations of ethanol.

*Paraffin-embedded fixed tissues are dewaxed in xylene before protease and formaldehyde treatment.
vProbes are available commercially
v both probe and target must be denatured prior to hybrid- ization.
v FISH slides should not be subjected to prolonged light exposure.
§ allows analysis of small regions
not visible by regular
chromosome banding.
§ Probes that cover the entire
chromosome, or whole
chromosome paints, are valuable
for detecting these small or
complex rear- rangements
§ spectral karyotyping can
distinguish all 23 chromosomes
by chromosome-specific colors.2
The culture of cells for 72 hours.

45 minutes before harvesting, colcemid is added to the cultures to arrest dividing cells in metaphase.

Cells are then suspended in a hypotonic medium (0.075 M KCl) and fixed with methanol/acetic acid (3:1).

fixed-cell suspension is applied to an inclined slide and allowed to dry.

second treatment with 70% acetic acid

improve the chromosome spreading and decrease background noise

Once the slide is dried, hybridization proceeds

§ Intrachromosomal amplifications or deletions can be detected

§ DNA from test and reference samples is labeled and used as a probe on a normal metaphase chromosome spread

§ One advantage of CGH is its capability to identify the location of deletions or amplifications throughout the genome

§ the test DNA is isolated and labeled along with a reference DNA.

§ Two colorimetrically distinct cyanine dyes, commonly Cy3 and Cy5, are used as fluorescent labels for the test and reference
DNA.
§ Cy3 = 550nm, green
§ Cy5 = 650 to 667 nm , red
Separate aliquots of test and reference DNA are labeled with different Cy3 and Cy5 dyes,

Application to a normal meta- phase spread

Test DNA is partially digested with DNase

to produce fragments that will bind efficiently to the de- natured DNA in a metaphase
chromosome spread.
v Although CGH requires advanced technical expertise, it has shown value in identifying recurrent
genomic im- balances not detected by karyotyping.