Week 4 - Cytogenetics Activity 3 Flashcards
FISH Principles
DNA probes bind to specific target sequences
Probes labelled with fluorescent dyes
Hybridisation process: single stranded DNA anneals to complementary DNA
Hybridisation of the probe to target DNA
- visualised as a brightly coloured signal by fluorescence microscopy
What can you use FISH on?
Metaphase chromosomes
- interphase cells
Cytogenetic preparations from cultured bone marrow, peripheral blood or lymph node tissue
Direct bone marrow or peripheral blood smears/cytospins
Fixed and sectioned tissue (paraffin-embedded)
Direct and Indirect Labelling of the Test Probe
Direct
- incorporating a fluorochrome-conjugated nucleotide into the probe e.g. fluorescein dUTP
Indirect
- probe labelled with reporter molecules such as biotin or digoxygenin
- these are detected by adding fluorescein conjugated avidin or anti-digoxygenin after hybridisation
Probes for FISH
Probes that target:
- Multiple chromosomal sequences
- Specific chromosome structure
- Unique DNA sequences
Probes for FISH - Probes that Hybridise to Multiple Chromosomal Sequences
Whole chromosome paints (WCP)
- for the entire chromosome
Available for each of the human chromosomes
Utilise cloned DNA libraries derived from whole flow-sorted human chromosomes
Useful for:
- resolving complex changes
- marker/ring chromosomes
- investigating/confirming abnormalities (structural abnormalities with additional unknown material)
Probes for FISH - Probes that Identify a Specific Chromosome Structure
Centromeric probes (CEP)
Highly repetitive human satellite DNA sequences
- α- and β- satellite sequences
Useful for:
- enumeration of chromosomes
- in a normal diploid cell: 2 hybridisation signals
- suitable for the detection of aneuploidy e.g. monosomies, trisomy’s etc.
Probes for FISH - Probes that Hybridise to Unique DNA Sequences
Locus specific indicators (LSI)
Genomic clones target specific genes or unique DNA sequences
Useful for detecting structural rearrangements e.g. deletions, translocations, inversions, variants
Application now:
- combine different probes -> multicolour
- FISH allows for the detection of several target sequences simultaneously
- e.g. dual colour dual fusion translocation probes, break-apart probes
FISH Strategies for the Detection of Recurring Translocations - Dual Colour, Dual Fusion Translocation Probes
Designed to detect gene fusions in recurring translocations
The probes span the breakpoints in the genes involved
Sensitivity - lower rate of false-positive cells cut-off for false-pos is ~1%
FISH Strategies for the Detection of Recurring Translocations - Dual Colour, Break-Apart Probes
DNA sequences from the 5’ and 3’ regions of a single gene are differentially labelled and detected with red and green fluorochromes
Sensitivity - high ~2%
Excellent specificity
E.g. IGH@ breakapart probe used when its uncertain which partner chros is involved
Advantages of FISH
Rapid, 4hr hybridisation Increased number of cells can be scored in a short time Efficiency of hybridisation & detection is increased Sensitivity & specificity is increased Can be used on: - non dividing cells - poor samples with too few cells - poor quality chromosomes
Limitations of FISH
Data can be obtained only for the target chromosomes
FISH is not a good screening tool for cytogenetically heterogeneous diseases e.g. AML or ALL
Restricted to those abnormalities that can be detected with currently available commercial probes
Only one or a few abnormalities can be assessed simultaneously
Probes used for MM Case Studies in FISH
Hyperdiploidy 5,9 and 15 t(14:16) - IGH:MAF t(4:14) - IGH:FGFR3 t(11:14) - IGH:CCND1 1p deletion - CDKN2C 1q amplification - CKS1B
Probes used for APML Case Studies in FISH
Karyotype
PML-RARA dual colour, dual fusion translocation probe
