2. 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

3. 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

4. Molecular Cytogenetic Techniques Powerful complement to conventional cytogenetic analysis of:
aneuploidy
structural rearrangements
submicroscopic rearrangements
microdeletions/duplications
subtelomere rearrangements

5. Basic chromosomal analysis
Patient
Basic chromosomal analysis
Family of the patient
Molecular cytogenetic analysis
Molecular biological analysis

6. 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)
hybridization
PCR

7. 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

8. Molecular Cytogenetic testing
POSTNATAL   Stat Blood   Routine Blood   Skin Biopsy   Product of Conception

9. PRENATAL   Amniotic Fluid   Chorionic Villus Sampling   Fetal Cord Blood
CANCER GENETICS   Bone Marrow   Oncology Blood   Solid Tumor   Lymph Node   Pleural Effusion   Core Biopsy

10. Molecular Application
FISH
CGH
PCR
Real Time PCR
DNA Sequencing
Microarray

11. Fluorescence In Situ Hybridization (FISH)

12. 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.

13. (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

14. FISH for Detection of Single to Multiple Genetic Events
Dual Targets
Two colors
Multiple
Targets
Multi- colors
Single Target
One color
Allows one to look at multiple genomic changes within a
single cell, without destruction of the cellular morphology.

15. 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

16. Probes Types of labeling Direct & Indirect
Radioactive (32P, 35S, 14C, 3H)
Fluorescent
FISH: fluorescent in situ hybridization
Biotinylated (avidin-streptavidin)

17. 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)

18. DIRECT FLUORESCENT -LABELED PROBE
Specimen DNA F T A A T C G T A G A
COVALENT BOND G C T C F
FISH Probe DNA

20. Types of FISH Probes
Centromere
Telomere
Whole chromosome paint
locus

21. Types of probes Centromeric (satellite) probes Locus specific probes
Whole chromosome painting probes

22. 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

23. Types of probes LSI Locus Specific Identifiers Deletion Probes
Translocation Probes
Gene Detection & Localization
Gene Amplification Probes

24. 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

25. 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

26. 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.

28. FISH Procedure Denature the chromosomes Denature the probe
Hybridization
Fluorescence staining
Examine slides or store in the dark

29. FISH Procedure

30. Direct Label FISH Technology18

31. Hybridization
target DNA
denaturation
hybridization
probe

32. 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

33. Automated Hybridization
HYBrite™
The probe and target DNA are denatured together.
Faster, easier, and safer hybridization.

35. 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.

36. Fluorescent Microscope
CCD Camera
Fluorescent Microscope
FISH Analysis
Software
Filters

38. FISH vs. Karyotyping 99.9% correlation Results:  24 hours
X (green), Y (red)
18 (aqua)
13 (green)
21 (red)
99.9% correlation
Results:  24 hours
Results: days 20