Presentation on theme: "Applications of Molecular Cytogenetics Dr Mohammed Alqahtani CSLT(CG), CLSp(CG), RT,MBA, Ph.D Genomic Medicine Unit Founder & Director Center of Excellence."— Presentation transcript:
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
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) hybridization PCR
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
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 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 ( 32 P, 35 S, 14 C, 3 H) 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 A A G G G G C C T T A A T T T T C C C C G G A A T T A A COVALENT BOND F F F F 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
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 (red) 18 (aqua) 13 (green) 21 (red) 99.9% correlation Results: 24 hours Results: days