2 Applications of Molecular Cytogenetics Dr Mohammed AlqahtaniCSLT(CG), CLSp(CG), RT,MBA, Ph.DGenomic Medicine Unit Founder & DirectorCenter of Excellence in Genomic Medicine Research Founder & Director
3 Lecture ObjectivesUnderstand how molecular cytogenetic techniques can be used to identify clinically relevant chromosome abnormalitiesBe aware of the different types of molecular techniques that can be used to identify and clarify chromosome rearrangements
5 Basic chromosomal analysis PatientBasic chromosomal analysisFamily of the patientMolecular cytogenetic analysisMolecular 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 situquantitative fluorescent PCR, real time PCRmethods based on amplification of probe attached to target sequence (MLPA, MAPH)hybridizationPCR
12 FISHA 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.
14 FISH for Detection of Single to Multiple Genetic Events Dual TargetsTwo colorsMultipleTargetsMulti- colorsSingle TargetOne colorAllows one to look at multiple genomic changes within asingle cell, without destruction of the cellular morphology.
15 ProbesProbe is a nucleic acid thatcan be labeled with a marker which allows identification and quantitationwill hybridize to another nucleic acid on the basis of base complementarity
16 Probes Types of labeling Direct & Indirect Radioactive (32P, 35S, 14C, 3H)FluorescentFISH: fluorescent in situ hybridizationBiotinylated (avidin-streptavidin)
17 ProbeA 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), YACPCR-product (amplification of certain segment of chromosomal DNA)
18 DIRECT FLUORESCENT -LABELED PROBE Specimen DNAFTAATCGTAGACOVALENT BONDGCTCFFISH Probe DNA
20 Types of FISH ProbesCentromereTelomereWhole chromosome paintlocus
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 chromosomeCEP Chromosome Enumerator Probes (centromere area)Most are Alpha and Satellite III ProbesCentromere regions stained brighter - means they are rich in A-T bonds
23 Types of probes LSI Locus Specific Identifiers Deletion Probes Translocation ProbesGene Detection & LocalizationGene Amplification Probes
24 In which conditions we have to indicate FISH analysis? The material doesn't contain metaphase chromosomesUnsuccessful cultivationIt 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 chromosomesAnalysis of low-frequency mosaicDiagnosis of submicroscopic (cryptic) chromosomal rearrangementsMicrodeletion syndromesAmplification of oncogenes and microdeletion of tumor-suppressor genes in malignancies
26 Multi Color FISHMulticolor 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.
32 HybridizationNucleic acid hybridization is the formation of a duplex between two complementary sequencesIntermolecular hybridization: between two polynucleotide chains which have complementary basesDNA-DNADNA-RNARNA-RNAAnnealing 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 processThe molecule emits a particular color when viewed through a fluorescence microscope that is equipped with the appropriate filter sets.