Fluorescence In-Situ Hybridisation (FISH)

Fluorescence In-Situ Hybridisation (FISH)

Molecular cytogenetic technique

Detect and localise specific DNA sequences

Fundamental principles of FISH

-Use of fluorescentyl labelled DNA probes

-Binding to complementary target sequences

-Visualisation of genetic loci and chromosomal abnormalities

Origins of FISH

in 1980s

Conceptualised as a molecular biology technique

Study of DNA sequence organisation and distribution within chromosomes

Prominence of FISH in cytogenetics and molecular diagnostics

Accurate identification of genetic aberrations and arrangements

Detection of gene copy number variations at cellular level

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

Critical for FISh success

Short, single-stranded DNA sequences labelled with fluorescent dyes

Selection considerations

-Specificity to target DNA sequences

-Probe length

-GC content

-Avoidance of repetitive DNA elements

Types of probes

-Whole chromosome painting probes

-Locus-specific probes

-Centromere-specific probes

-Utilised in various FISH applications for visualising genetic loci

Essentiality of sample preparation

Critical for successful FISh analysis

Involves collection and fixation of biological specimens

Fixation methods of sample

-Chemical fixation eg formaldehyde, methanol

-Immobilisation and stabilisation of cellular components

-Enables subsequent probe hybridisation and imaging

Hybridisation process in FISH

Denaturation of target DNA

Probe binding, annealing and incubation

Under controlled conditions

Direct Detection method

Direct detection=fluorescently labelled probed

Indirect detection method

Amplification techniques

eg tyramide signal amplification (TSA)

Enhances signal and improves detection sensitivity

Critical role of imaging and microscopy

Enable visualisation and analysis of fluorescently labelled DNA probes bound to target DNA sequences

Equipment used for FISH analysis

-High resolution fluorescence microscopes

-Appropriate filter sets

-Imaging software

Functionality of FISH analysis

Capture fluorescent signals emitted by probes

Visualise chromosomal or genetic abnormalities at sub-cellular level

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)

What is the importance of prenatal screening using FISH?

Allows timely and accurate prenatal diagnosis

Aids informed decision-making and appropriate prenatal care

Detection of genetic markers in cancer using FISH

Visulaise genetic markers eg:

-HER2/neu amplification in breast cancer

-BCR-ABL fusion in chronic myeloid leukaemia

-MYC amplification in solid tumours

Understanding FISH patterns and signals

Comprehending patterns and signals related to:

-Number

-Size

-Location

Correspond to DNA probes bound to specific chromosomal or genetic loci

Examples of patterns in FISH

-Diploid signals

-Monosomy

-Trisomy

-Deletion

-Duplication

-Translocation

-Split signals

Down Syndrome Diagnosis

Maternal blood tested or ultrasound finding can lead to genetic testing

Foetal DNA karyotyping or FISH used

Detection of Chromosomal Aberrations

Numerical chromosmal aberrations in formalin-fixed foetal tissue

Karyotype in cases wth sufficient cells and diagnosed numerical aberrations in foetal malformations

Clinical significance and implications

Used for accurate disease diagnosis, prognosis and treatment decisions

Nuremberg Code

Emphasise voluntary consent and minimising risks in clinical research studies

Declaration of Helsinki

Provides ethical principle for biomedical research

Emphasise the protection of research participants

Belmont report

Outlines principle of respect for persons, beneficence and justice

Serve as foundation for ethical conduct in research practices

Confidentiality

Laws such as HIPAA

Protect patients health information and require consent for disclosure

Informed consent

Principles of voluntary conset and provision of relevant information to patients

Fundamental in ethical conduct of FISH testing and research

HIPAA

Sets limitations on disclosure of protected health information and established requirements for consent in USA

State and Federal regulations

Specific regulations and codes governing the handling of health information, reporting abuse cases and requirement for informed consent. in UK and USA

Ensure ethical conduct

Future directions for FISH

-High-resolution Imaging

-Multiplex FISH

-Microfluidic Plastforms

High resolution imaging and FISH

Integration of super resolution imaging systems for enhanced visualisation of nuclear structures and gene functions

Multiplex FISH

Probe labelling efficiency faciliating development of this assay

Detect multiple DNA or RNA sequences in a single cell

Microfluidic platforms

Streamline process

Offer miniaturised and automated solutions for detecting chromosome abnormalities

Personalised medicine

Increasing demand for FISH

Contribute to tailored therapies in cancer and other diseases

Single-Cell Analysis

Single-molecule RNA FISh and RNA-DNA Fish

New avenue for quantitative imaging of RNA molecules

Simultaneous detection of mRNA and protein in single cells

Expanded disease research

FISH playing a pivotal role in detecting repeats in human diseases, analyse sperm aneuploidy frequencies

Contribute to diagnosis and tailored therapies in solid tumours