Cytogenetics Lesson 10 FISH

7/30/2019 Cytogenetics Lesson 10 FISH

1/27

Meaning


2/27

Uses


3/27

MethodSAMPLE PREPARATIONCultured or uncultured lymphocytes are fixed onto a microscope slide.Slide is soaked in SSCSlide is dehydrated in alcohol

Probe is added to the slideProbe area is sealed with a coverslip

DENATURATIONHeat slide to 800C for 5 minutesThe double strands of sample and probe DNA break apart (denature)

HYBRIDISATIONIncubate slide overnight at 370CSpecific probe region will bind to corresponding DNA region

POST HYBRIDISATIONSoak slides in SSC at 640C for 2 minutes - removes non specific, looselybound DNA

Rinse slides in alcoholAdd Dapi/Antifade solutionSeal with a coverslipMICROSCOPYAnalyse metaphases for presence of control regionAnalyse relevant chromosome for probe presence/absenceCount nuclei, noting deletion status


4/27

Temperature controlled slide

processing system


5/27

Slide

Technologist The relevant

excitation filter is

used depending on

the specificwavelength of the

fluorophore being

used. This will

excite the dye on

the slide whichwill emit light that

is filtered by the

emission filter to

give a

homogeneouscolour for each

probe being

examined


6/27

Aneuploidy FISHAnalysis for Aneuploidy


7/27

Aneuploidy

Turner syndrome

Aneuploidy Fish


8/27

Aneuploidy


9/27

Aneuploidy


10/27

Aneuploidy


11/27

No Deletion Found (2 green control signals; 2 red DNA syndrome probe signals)

Deletion Detected (2 green control signals; 1 red DNA syndrome probe signals)

Microdeletion Probes


12/27

Di George

Clinical Symptomsinclude one or a combination of the following:congenital heart disease (particularly conotruncal malformations)palatal abnormalities [especially velopharyngeal insufficiency (VPI)]hypocalcemiaimmune deficiencylearning difficultiescharacteristic facial featuresDiagnosis

FISH detection of a 22q11.2 deletion in the DiGeorge chromosomal region(DGCR).Cytogenetic studies for the presence of a translocation involving 22q11.2(1%)

Deletion 10p13-p14.Mode of InheritanceThe 22q11.2 deletion is inherited in an autosomal dominant manner.About 93% of probands have a de novo deletion of 22q11.2.7% inherited the 22q11.2 deletion from a parent.Both parents of an individual with 22q11.2 deletion should have FISH testingGermline mosaicism

del 22q11.2.Includes:

DiGeorge Syndrome (DGS)

VelocardiofacialSyndrome (VCFS)

Shprintzen Syndrome

Conotruncal Anomaly

Face Syndrome (CTAF)

Caylor Cardiofacial

Syndrome

Autosomal DominantOpitz G/BBB Syndrome

22q11.2 Deletion Syndrome


13/27

22q11.2 Deletion Syndrome


14/27

Microdeletion 15q12


15/27

Microdeletion 15q12Deletion on Paternal Chromosome 15 leads to PW syndrome.

Deletion on Maternal chromosome 15 leads to Angelman Syndrome.


16/27

Microdeletion 15q12


17/27

Whole chromosome PaintUSES:TRANSLOCATIONS


18/27

USES:TRANSLOCATIONS


19/27

USES:TRANSLOCATIONS


20/27

USES:TRANSLOCATIONS


21/27

Preimplantation Genetics


22/27



23/27



24/27

USES: ONCOLOGY


25/27

USES: ONCOLOGY


26/27

USES: ONCOLOGY


27/27

Software measures HER2 amplification in breast tumor. For eachcell the software reports HER2 & CEP17 copy numbers and the

HER2:CEP17 ratio.

Documents

Cytogenetics Lesson 10 FISH