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
Molecular cytogenetic analysis
Family of the patient
Molecular biological analysis
Molecular cytogenetic examinations
• 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)
hybridizationPCR
Molecular Cytogenetics Era
• 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
• FISH
• CGH
• PCR
• Real Time PCR
• DNA Sequencing
• Microarray
Fluorescence In Situ Hybridization (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
(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 FLUORESCEN T - LABELED PROBE
AA GG GG CC TT AA TT
TT CC CC GG AA TT AA
COVALENT BOND
FF
FF
Specimen DNA
FISH Probe DNA
Types of FISH Probes
• Centromere
• Telomere
• Whole chromosome paint
• locus
Types of probes
Centromeric (satellite) probes
Locus specific probes
Whole chromosome painting 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
Types of 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
• 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
In which conditions we have to indicate FISH analysis
?
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
• Denature the chromosomes
• Denature the probe
• Hybridization
• Fluorescence staining
• Examine slides or store in the
dark
FISH Procedure
Direct Label FISH Technology
Direct Label FISH Technology
Hybridization
target DNA
probe
denaturation
hybridization
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.
Fluorescent Microscope
CCD Camera
Filters FISH Analysis
Software
FISH vs. Karyotyping
X (green), Y X (green), Y (red)
(red) 18 (aqua) 18 (aqua)
13 (green) 13 (green) 21 (red) 21 (red)
99.9% correlation 99.9% correlation
Results:
Results: 24 hours 24 hours Results: 7 - 10 daysResults: 7 - 10 days
