Transcript paint software download

Applications of Molecular
Cytogenetics
Dr Mohammed Alqahtani
CSLT(CG), CLSp(CG), RT,MBA, Ph.D
Genomic Medicine Unit Founder & Director
Center of Excellence in Genomic Medicine Research
Founder & Director
Lecture Objectives
• Understand how molecular cytogenetic
techniques can be used to identify
clinically relevant chromosome
abnormalities
• Be aware of the different types of
molecular techniques that can be used to
identify and clarify chromosome
rearrangements
• Molecular Cytogenetic Techniques
Powerful complement to conventional
cytogenetic analysis of:
– aneuploidy
– structural rearrangements
– submicroscopic rearrangements
• microdeletions/duplications
• subtelomere rearrangements
Patient
Basic chromosomal analysis
Family of
the patient
Molecular cytogenetic analysis
Molecular biological analysis
Molecular cytogenetic examinations
PCR
hybridization
• In most of cases interphase cells could be used
for analysis (with exception of whole chromosome
painting probes and M-FISH)
• Examples of methods:
– in situ hybridization and its modifications (CGH, M-FISH,
fiber FISH atd.)
– Gene chips, resp. array CGH, DNA microarray etc.
– PRINS, PCR in situ
– quantitative fluorescent PCR, real time PCR
– methods based on amplification of probe attached to
target sequence (MLPA, MAPH)
Molecular Cytogenetics Era
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1988 FISH
1992 Comparative Genomic Hybridization
1994 Reverse FISH
1996 Spectral Karyotyping, M-FISH
1999 M-Band analysis
2002 Fiber FISH
2002 Primed in situ labeling (PRINS)
2002 Microarray
Molecular Cytogenetic testing
• POSTNATAL
Stat Blood
Routine Blood
Skin Biopsy
Product of Conception
• PRENATAL
Amniotic Fluid
Chorionic Villus Sampling
Fetal Cord Blood
• CANCER GENETICS
Bone Marrow
Oncology Blood
Solid Tumor
Lymph Node
Pleural Effusion
Core Biopsy
Molecular Application
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FISH
CGH
PCR
Real Time PCR
DNA Sequencing
Microarray
Fluorescence In Situ Hybridization
(FISH)
FISH
• A technique that hybridizes a DNA nucleic acid
probe to a target DNA sequence contained
within a cell nucleus.
• A variety of specimen types can by analyzed
using FISH. The intact cells are attached to a
microscope slide using standard cytogenetic
methods.
(FISH) TO RULE OUT:
Chromosome Microdeletion Detection
Interphase Chromosome Enumeration
Gene Rearrangements (ie, bcr/abl,
PML/RARA)
Cryptic Chromosomal Rearrangements
Marker Chromosome Identification
Chromosome Breakpoint Mapping
FISH for Detection of Single to Multiple Genetic Events
Single Target
One color
Dual Targets
Two colors
Multiple
Targets
Multi- colors
Allows one to look at multiple genomic changes within a
single cell, without destruction of the cellular morphology.
Probes
• Probe is a nucleic acid that
– can be labeled with a marker which
allows identification and quantitation
– will hybridize to another nucleic acid
on the basis of base complementarity
Probes
Types of labeling
• Direct & Indirect
• Radioactive (32P, 35S, 14C, 3H)
• Fluorescent
• FISH: fluorescent in situ
hybridization
• Biotinylated (avidin-streptavidin)
Probe
• A part of DNA (or RNA) that is complementary to
certain sequence on target DNA (i.e. DNA of the
patient)
• Plasmid, phage DNA, cosmid (or combination of
phage and plasmid DNA), YAC
• PCR-product (amplification of certain segment of
chromosomal DNA)

DIRECT FLUORESCENT LABELED PROBE
Specimen DNA
F
T
A
A
T
C
G
G
C
A
C
A
T
T
COVALENT BOND
G
F
FISH Probe DNA
Types of FISH Probes
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Centromere
Telomere
Whole chromosome paint
locus
Types of probes
Centromeric (satellite)
probes
Locus specific probes
Whole chromosome painting probes
Types of probes
• Telomeric probes
have specificity for a single human chromosome
arm. They contain a locus estimated to be within
300 kb of the end of the chromosome.
• WCP Chromosome Painting Probes
the hybridized probe fluoresces with bright intensity
along the length of chromosome
• CEP Chromosome Enumerator Probes
(centromere area)
– Most are Alpha and Satellite III Probes
– Centromere regions stained brighter - means they
are rich in A-T bonds
Types of probes
• LSI Locus Specific Identifiers
– Deletion Probes
– Translocation Probes
– Gene Detection & Localization
– Gene Amplification Probes
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
In which conditions we have to
indicate FISH analysis?
• Identification of marker chromosomes
• Analysis of low-frequency mosaic
• Diagnosis of submicroscopic (cryptic)
chromosomal rearrangements
– Microdeletion syndromes
– Amplification of oncogenes and microdeletion
of tumor-suppressor genes in malignancies
Multi Color FISH
• Multicolor FISH can provide “colorized”
information relative to chromosome
rearrangements, especially useful in
specimens where chromosome
preparations are less than optimal for
standard cytogenetic banding analysis.
FISH Procedure
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Denature the chromosomes
Denature the probe
Hybridization
Fluorescence staining
Examine slides or store in the
dark
FISH Procedure
Direct Label FISH Technology
Hybridization
target DNA
denaturation
hybridization
probe
Hybridization
• Nucleic acid hybridization is the formation of a
duplex between two complementary sequences
• Intermolecular hybridization: between two
polynucleotide chains which have complementary
bases
– DNA-DNA
– DNA-RNA
– RNA-RNA
• Annealing is another term used to describe the
hybridization of two complementary molecules
Automated Hybridization
HYBrite™
• The probe and target
DNA are denatured
together.
• Faster, easier, and
safer hybridization.
Visualization of the Probe
• DNA probe is labeled with a colored fluorescent
molecule.
• This fluorescent molecule remains attached to
the DNA during the hybridization process
• The molecule emits a particular color when
viewed through a fluorescence microscope that
is equipped with the appropriate filter sets.
CCD Camera
Fluorescent Microscope
Filters
FISH Analysis
Software
FISH vs. Karyotyping
13 (green)
21 (red)
99.9% correlation
X (green), Y (red)
18 (aqua)
Results:  24 hours
Results: 7 - 10 days