Applications of MolecularCytogenetics

Dr Mohammed AlqahtaniCSLT(CG), CLSp(CG), RT,MBA, Ph.D

Genomic Medicine Unit Founder & DirectorCenter 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)

hybr

idiz

atio

nP

CR

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 DetectionInterphase Chromosome EnumerationGene Rearrangements (ie, bcr/abl,

PML/RARA)Cryptic Chromosomal Rearrangements Marker Chromosome IdentificationChromosome 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 asingle 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 FLUORESCENT -LABELED PROBE

AA GG GG CCTT

AATT

TT CC CC GGAA

TTAA

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 TechnologyDirect 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

FiltersFISH 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% correlation99.9% correlation

Results: Results: 24 hours 24 hours Results: 7 - 10 daysResults: 7 - 10 days