Download Chromosomes Identification

Chromosomes Identification
Definitions
• Cytogenetics
– Involves visual study of chromosomes at
microscopic level
– Both the number and structure of the
chromosomes are analyzed.
• Karyotype
– Chromosome complement
– Also applied to picture of chromosomes
Preparation of Metaphase Chromosomes
for Analysis
• For diagnostic purposes, chromosomes are usually analyzed in
metaphases prepared from cell cultures
• Cell source:
- Blood cells
- Skin fibroblasts
- Amniotic cells / chorionic villi
• Chromosome analysis from blood :
• Five principal steps are required:
1- lymphocyte culture
2- harvest of metaphase chromosomes
3- chromosome preparation
4- staining the chromosomes
5- analysis by microscopy, nowadays assisted by computer analysis.
Preparation of Metaphase Chromosomes
for Analysis cont….
• For a lymphocyte cell culture, either peripheral blood is used directly or
lymphocytes are isolated from peripheral blood (T lymphocytes).
• A sample of about 0.5mL of peripheral blood is needed.
• Heparin is must be added to prevent clotting, (The proportion of heparin
to blood is about 1: 20 )
• A special culture media solution is used , phytohemoagglutinine is added
to stimulate the division .
• A lymphocyte culture requires about 72 hours at 37C for two cell divisions.
• Cells reaching mitosis are arrested in metaphase by adding a suitable
concentration of a colchicine derivative (colcemide) for two hours prior to
harvest.
• Colcemide interferes with spindle formation and thus arrests mitosis in
metaphase, yielding a relative enrichment of cells in metaphase.
• About 5% of cells will be in mitosis after 72 hours.
• The culture is then terminated and the cells in metaphase are harvested
Preparation of Metaphase Chromosomes for
Analysis cont….
• At harvest, the culture solution is centrifuged
• Hypotonic potassium chloride solution
• (KCl, 0.075 molar) is added to the collected cells for 20 minutes , then
cetrifugation .
• A fixative solution of a 3:1 mixture of methyl alcohol and glacial acetic
acid is added .
• Usually the fixative is changed 4–6 times with subsequent centrifugation.
• The fixed cells are taken up in a pipette, dropped onto a clean glass slide
suitable for microscopic analysis, and air-dried
• The preparation is treated according to the type of bands desired , stained
and the slide is covered with a cover glass and examined under
microscope
• between 20 and 100 metaphases are examined. Some of the metaphases
• are photographed under the microscope and subsequently can be cut out
from the photograph (karyotyping).
Chromosomal Analysis from the Blood
Staining techniques
• There are to types of staining techniques :
1- Solid staining .
2- Banding techniques .
Solid Staining
• Stain the chromosomes uniformly (one color).
• Different chromosomes can be parially
differentiated .
• C group ( chr. 6 → 12 ) hard to differentiate
• Structural chromosomal abnormalities can not
be detected .
• It used now to detect chromosomal fragile
sites eg. Fragile X chromosome .
Solid Staining
Feulgen Staining:
- Cells are subjected to a mild hydrolysis in 1N HCl
at 600C for 10 minutes.
- This treatment produces a free aldehyde group
in deoxyribose molecules.
- Then Schiff’s reagent is used ,it gives a deep
pink colour.
- Ribose of RNA will not form an aldehyde under
these conditions, and the reaction is thus specific
for DNA
Solid Staining of Mitotic
Chromosomal Spread
Schiff’s reagent stain
Chromosomal Banding Techniques
• Several techniques have been developed for inducing
specific patterns of light and dark transverse bands along
each metaphase chromosome: the banding patterns,
which can be visualized under the microscope
• Each chromosomes can be identified by its banding
pattern
• The heterochromatin regions in a chromosome distinctly
differ in their stain ability from euchromatic region.
Banding techniques
•
•
•
•
•
G banding
Q banding
R banding
C banding
Ag-NOR stain
(active)
- Giemsa
- Quinacrine
- Reverse ( Giemsa )
- Centromeric (heterochromatin)
- Nucleolar Organizing Regions
G banding
• Most common method used
• Many techniques are available, each involving some pretreatment of the
chromosomes .
• In ASG (Acid-Saline-Giemsa) cells are incubated in citric acid and NaCl for
one hour at 600C and are then treated with the Giemsa stain
• Or chromosomes is treated with trypsin (denatures protein ) before
staining .
• Giemsa stain (preferentially stain AT rich regions)
– Each individual chromosome has a characteristic light and dark bands
– 400 bands per haploid genome
– Each band corresponds to 5-10 mega bases
– High resolution (800 bands ; prometaphase chromosome)
– use methotrexate and colchicine
• Dark bands are gene poor ( AT rich , heterochromatin )
• Dark bands are SAR (Scaffold Attachement Regions) rich .
Normal male karyotype with G
banding
Q banding
* The Q bands are the fluorescent bands detected
after quinacrine mustard staining .
* Examined under fluorescent microscope.
* Preferentially stain AT rich regions .
* Similar pattern to G banding .
* Used especially for Y chromosome abnormalities
or mosaicism .
* The Y chromosome become brightly fluorescent
both in the interphase and in metaphase.
R banding
• R (reverse banding)
preferentially stains GCrich regions .
• Chromosomes are
heated before staining
with Giemsa
• Light and dark bands
are reversed
• Dark bands are gene
rich ( euchromatin )
• Dark bands are SAR
(Scaffold Attachement
Regions) poor
C banding
•
•
Used to identify centromeres / heterochromatin
Heterochromatic regions
–
–
–
•
contain repetitive sequences
highly condensed chromatin fibres
preferentially stains constitutive heterochromatin, found
in the centromere regions and distal Yq .
Treat the chromosomes with( denaturation & staining)
1.
2.
3.
Acid (denaturation )
Alkali (denaturation )
Then G stain (staining )
C-banded karyotype of XY cell
Idiogram
ISCN
• International System for
Human Cytogenetic
Nomenclature ( ISCN )
• Each area of chromosome
given number
• Lowest number closest
(proximal) to centromere
• Highest number at tips
(distal) to centromere
• ARM
REGION
BAND
S
ub-BAND, numbering from
the centromere progressing
distal
ISCN
•
•
•
•
•
•
•
del
dic
fra
i
inv
p
r
- deletion
- dicentric
- fragile site
- isochromosome
- inversion
- short arm
- ring
•
•
•
•
•
•
•
der - derivative
dup - duplication
h - heterochromatin
ins - insertion
mat - maternal origin
q - long arm
t - translocation
ISCN
46,XX,del(5p)
• , separates
– chromosome numbers
– sex chromosomes
– chromosome abnormalities
;
46,XX,t(2;4)(q21;q21)
• ; separates
– altered chromosomes
– break points in structural rearrangements involving
more than 1 chromosome
Two kinds of cytogenetic examination:
• Basic chromosomal analysis
– staining methods (solid staining, banding staining )
– Based on analysis of metaphase chromosomes
• Molecular cytogenetic analysis
– Identification of chromosomal abnormalities using
molecular biological methods
Fluorescence in-Situ Hybridization
(FISH)
•
•
•
•
•
•
•
•
FISH applies molecular genetic techniques to chromosome preparations in
metaphase or interphase nuclei, an approach called molecularcytogenetics.
The aim is to to map genes and to detect small chromosomal rearrangements that
cannot be detected by microscopy .
Conventional chromosomal analysis can detect the loss or gain of chromosomal
material of 4 million base pairs (4 Mb) or more.
In FISH a labeled DNA probe is hybridized in situ to single-stranded chromosomal
DNA on a microscope slide.
Site-specific hybridization results in a signal visualized over the chromosome.
A distinction is made between direct and indirect nonisotopic labeling.
In direct labeling the fluorescent label, a modified nucleotide (often 2
deoxyuridine 5 triphosphate) containing a fluorophore, is directly incorporated
into DNA.
Indirect labeling requires labeling the DNA probe with a fluorophore to make the
signal visible.
Principle of FISH
• With indirect nonisotopic labeling, metaphase or interphase
cells fixed on a slide are denatured into single-stranded DNA .
• A DNA probe is labeled with biotin and hybridized in situ to
its specific site on the chromosome .
• This site is visualized by fluorescence in dark field microscopy
by binding a fluorescent-dye labeled antibody (for biotin this
antibody is streptavidin) to the biotin .
• This is the primary antibody.
• To enhance the intensity of fluorescence, a secondary
antibody (here, a biotinylatedanti-avidin antibody) is attached
• The resulting signal is amplified by attaching additional
labeled antibodies .
Hybridization
target DNA
denaturation
hybridization
probe
Probe : a part of DNA (or RNA) that is complementary to certain
sequence on target DNA
FISH (gene “probes”)
• Uses fluorescent labelled
DNA fragments, ~10,000
base pairs, to bind (or not
bind) to its complement
In which conditions we have to indicate
FISH analysis?
• The material doesn't contain metaphase chromosomes
– Unsuccessful cultivation
– It isn't possible to cultivate the tissue from patient (preimplantation
analysis, rapid prenatal examinations, examinations of solid tumors or
autopsy material)
•
•
•
•
Analysis of complicated chromosomal rearrangements
Identification of marker chromosomes
Analysis of low-frequency mosaic
Diagnosis of submicroscopic (cryptic) chromosomal
rearrangements (microdeletion syndromes )
Patient
Basic chromosomal analysis
Family of
the patient
Molecular cytogenetic analysis
Molecular biological analysis
Types of probes
Centromeric (satellite)
probes
Locus specific probes
Whole chromosome painting probes
Satellite (centromeric) probe
on X–chromosome
Possible karyotype?
45,X or 46,XY
X- and Y-centromeric probes
Green = X
Red = Y
Determine probable karyotype.
46,XY
Painting probes – examination of
chromosomes 1, 4 and 8
4
1
4
8
1
Normal finding
8
Can distinguish chromosomes by “painting ” – using DNA
hybridization + fluorescent probes – during mitosis