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Applications of Molecular Cytogenetics

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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)



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 Technology

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

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