Fluorescence In-Situ Hybridisation (FISH)

Fluorescence In-Situ Hybridisation (FISH)

Molecular cytogenetic technique
Detect and localise specific DNA sequences

High resolution 40-250 kb/probe

Fundamental principles of FISH

-Use of fluorescentyl labelled DNA probes
-Binding to complementary target sequences
-Visualisation of genetic loci and chromosomal abnormalities

Limitations

• Probe design requires knowledge of specific chromosomal Ab or region of interest to be studied

• Cutoff signal may be diff among labs

• Processing errors, imperfect hybridization, non-specific binding, photobleaching, inter-observer variability, and false positive and negative results are possible

Advantages

• Identify the presence and location of a region of DNA or RNA within morphologically preserved chromosome preparations, fixed cells or tissue sections

• Detect common numerical and structural Ab in uncultured cells

• Correlation between FISH result and tumor morphology with conventional light microscopy

• Allows simultaneous interrogation of mul cytogenetic targets

• Less labor-intensive method for confirming the presence of a DNA segment within an entire genome than other conventional methods like southern blotting

Evolution and advancements of FISH

-Improvement of probe design and fluorescence detection methods
-Integration with advanced imaging techniques
-Indispensable role in genetic research, clinical diagnostics and cancer biology

Role of FISH in medical science

-Precise detection and characterisation of genetic abnormalities
-Association with congenital and acquired diseases

Clinical diagnostics

identify chromosomal aberrations in prenatal and postnatal genetic testing
Diagnosis and prognosis of genetic disorders (eg Down syndrome, Turner syndrome and leukaemia)

Importance of FISH in cancer genetics

-Visualise gene amplifications, deletions and translocations in tumour cells
-Classify cancer subtypes, treatment selection and prognostic evaluation

Significance in genetic research

-Mapping DNA sequences to chromosomes
-Studying chromosomal architecture
-Elucidating the genetic basis of inherited diseases

DNA probe selection and design

Small DNA sequences labeled with fluorescent dye

Attach to another dna - can be genes, section of chro or whole chro, interphase cells

Type: spectrum orange, green, gold, aqua, red

Prepare from fragments of dna cloned into bacterial (BAC), P1 (PAC), yeast (YAC) —> BAC is the most common

Labeled directly or indirectly

Common fluorophores: fluorescein isothiocyanate (FITC) and Texas Red, green and red respectively

Selection considerations

-Specificity to target DNA sequences
-Probe length
-GC content
-Avoidance of repetitive DNA elements

Types of probes

-Whole chromosome painting probes

-Centromere-specific probes

-Locus-specific probes (LSI)

-Break-apart probes

-Fusion probes

-Telomeric probes

Whole chromosomal paint probes (WCP)

Hybridize to the entire length of one or more chro

→ isolating individual chro by flow cytometry or by chro microdissection and then PCR amplified with degenerate oligonucleotide primers

• cryptic translocation

• Marker chro

• Aneuploidy (count the num of chro)

Centromere probes (CEP)

Hybridize to repetitive dna sequences referred as alpha-satelitte dna near the centromere of the chro

Satellite dna located in centromeroc region of chro

Heterochro region of chro

• enumerate aneuploidy of specific chro in both meta and interphase

• Marker chro

• Centromeric examination

Locus-specific probes (LSI)

Hybridize to unique sequences ranged from 40-500 kb

• deletions (DGS, WBS, p53, p16)

• Duplication

• Translocation (BCR/ABL)

• Specific gene amplification in cancer (HER2 in breast cancer)

Break-apart probe

Adjunct regions are directly labeled with 2 did fluorophores

• normal cells: fusion signal

• Ab cells: separated signals

→ translocation

→ Structural rearrangements

Fusion probes

2 diff probes (usually on dif chro) are labeled with 2 diff fluorophore

• normal: 2 red 2 green

• Ab: fusion

→ translocation and structural rearrangements in cancer

Telomeric probes

Map telomeric sequences but to unique DNA sequences found very near the telomeres (called as subtellmeric probes)

Probes are available for 41 of 46 chromosomal telomeres

Interpretation of FISH result

Dipoidy nuclei: 2 dots

Duplication: > 2 dots

Loss: 1 or 0 dot

FISH procedure

• prepare and pretreat slide - met a phase, interphase or paraffin embedded slides

• Deraturing of probe DNA and Chr DNA, dehydrate slides

• Hybridization of the probe DNA and chr DNA overnight

• Post hybridization washing to remove unspecific and unbound probe

• Counterstaining and visualization using fluorescent microscope

Prepare slides

Metaphases and interphase are free of cytoplasm and debris

Using standard hypotonic and acid/methanol washes

Evaluate using a phase contrast microscope, chromosomes are full grey

100 cells/10 field

Store: -20oC up to 6 month, -80oC up to 1 year

Not recommend: bake the slides

Aging slides

→ sharper, avoid fiddised signals

Coupling jar 2cSSC (pH 7.0) at 37oC for 30 min

Fresh dropped: stored at room tem at least ONE day

More than 2 weeks: not be treated with aging method

Denaturing of probe DNA and chro DNA, dehydrate slides

Denaturing breaks the hydrogen bonds that hold the complementary strand of DNA together

72oC for 2-5min in 70% formamide/2xSSC (pH7.0) → lowers the melting point of DNA

Post washing

Remove unbound probe and probe cross-hybridized to non-specific regions of dna

→ time in solution wash affects signal strength

Reduce wash: inadequate to eliminate cross-hybridization (ko du De loai Bo Lai cheo)

Increase wash: reduce signal strength

Visualization using fluorescent mocroscope

The age of the bulb and its alignment can affect results

→ too old and not properly aligned: dark and weak signal

Counterstaining

Fluorescein isothiocyanate (FITC green) - propidium iodide (red)

Rodamine (red) or Texas Red - DAPI (blue)

More than one probe (red and green) - DAPI blue

Control in FISH

Internal control

• chromosome enumeration: 2 centromere probes in 2 chromosomes, each probe has did color

• microseletions: 2 probes in same chromosome, 1 with critical region, 1 outside the critical region

→ if a probe is used that does not produce an internal signal (ex Y in female sample), another sample having the probe target should be run in parallel

Troubleshooting - causes of inadequate hybridization (no or weak signal)

Covered by cytoplasm

Higher tem

Improper pH

Stringency of post wash is too high

Hybridization conditions not optimum

Inappropriate filter used

Troubleshooting - excessive background after dltetion (too much background)

Rewash with higher stringency

Slide not be cleaned prior to dropping cells

Cell pellet may contain too much debris → wash several times with fresh fixative

Rescheck the pH and tem of wash solution

Direct label

Indirect detection method

Amplification techniques
eg tyramide signal amplification (TSA)
Enhances signal and improves detection sensitivity

Equipment used for FISH analysis

-High resolution fluorescence microscopes
-Appropriate filter sets
-Imaging software

Genetic disease diagnosis using FISH

Detect chromosomal abnormalities, gene deletions or duplications
Identify genetic aberrations to conditions such as Down syndrome, Prader-Willi syndrome and Angelman syndrome

Prenatal screening using FISH

Used to detect common aneuploidies in foetal cells
Obtained through procedures like amniocentesis and chorionic villus sampling (CVS)

Primed in situ labeling PRINS

Short unlabeled dna primers to metaphase and subsequent in situ chain elongation catalyzed by a dna polymerase and dual color PRINS