o A powerful cytogenetic technique.

o It is used to detect localize the presence

or absence of specific DNA sequences on

chromosomes.

o Exploits the ability of single stranded

DNA to anneal to complementary DNA.

o Uses fluorescent probes.

o Fluorescence microscopy detects the

presence of fluorescent probes.

o It is a powerful technique used in the

detection of chromosomal abnormalities.

Fluorescence in situ hybridization (FISH) is a molecular

diagnostic technique utilizing labeled DNA probes to

detect or confirm gene or chromosome abnormalities.

FISH Targets

- Metaphase Chromosomes

- Interphase Nuclei

- Fixed Tissues

- Cells in culture

I t i s a r el at i vel y new cyt ogenet i c t echni que t hat al l ows a cyt ogenet i ci st t o det er mi ne how many copi es of a par t i cul ar chr omosome ar e pr esent wi t hout havi ng t o go t hr ough al l of t he st eps i nvol ved i n pr oduci ng a kar yot ype.For example,

FISH analysis can quickly tell you how many number 21 chromosomes

are present, but it cannot tell you anything about the structure of those

chromosomes.

How does FISH work?FISH is useful to help to identify where a particular gene

falls within an individual's chromosome.

A. The first step is to prepare short sequences of single-stranded

DNA that match a portion of the gene. These are called probes.

B. The next step is to label these probes by attaching one of a

number of colors of fluorescent dye.

C. DNA is composed of two strands of complementary molecules

that bind to each other like chemical magnets.

D. When a probe binds to a chromosome, its fluorescent tag

provides a way to see its location using fluorescent microscope.

General schematic diagram of FISH

Dire

ct a

nd

ind

irec

t lab

ellin

go

f pro

bes

DIRECT

FITC; rhodamine;Texas

red;cy2;cy3;cy5 and AMCA dyes

are most frequently used

INDIRECT

biotin;digoxigenin & DNP reprtr

molecules are frequently used

Tagging of probes by nick translation

Types of Probes

Locus specific probes bind to a

particular region of a chromosome.

This type of probe is useful when

scientists have isolated a small

portion of a gene and want to

determine on which chromosome

the gene is located.

Alphoid or centromeric repeat

probes are generated from

repetitive sequences found in

the middle of each

chromosome. Researchers use

these probes to determine

whether an individual has the

correct number of

chromosomes. These probes

can also be used in combination

with "locus specific probes" to

determine whether an individual

is missing genetic material from

a particular chromosome.

Whole chromosome

probes are actually collections

of smaller probes, each of

which binds to a different

sequence along the length of a

given chromosome. Using

multiple probes labeled with a

mixture of different fluorescent

dyes, scientists are able to

label each chromosome in its

own unique color. The resulting

full-color map of the

chromosome is known as a

spectral karyotype. Whole

chromosome probes are

particularly useful for

examining chromosomal

abnormalities, for example,

when a piece of one

chromosome is attached to the

end of another chromosome.

Chronic myeloid leukemia

• Cancer of White Blood Cells.

• Increased and unregulated groth of myeloid cells in

bone marow and accumulation of these cells in blood.

• It is a type of first malignancy to be linked to a clear

genetic abnormality which is the chromosomal

translocation known as philadelphia chromosome.

• More common in males.

Philadelphia chromosome

• In this translocation,

parts of chromosomes

9th and 22nd switch

places.

• As a result , part of

BCR gene from

chromosome 22 is

fused with ABL gene

on chromosome.

• BCR ABL fusion gene

prouct is a tyrosine

kinase-remains

continuously on.

• Green fluorescence – BCR gene

• Red fluorescence - ABL gene

• Yellow fluorescence – BCR ABL

fusion gene

Interphase FISH on a nucleus using an

Exta-signal probe to detect

the BCR/ABL translocation.The green

signal indicates the presence of

the BCR gene, red signals indicate the

presence of the ABL gene and the red-

green fusion (yellow) signal confirms a

BCR/ABL translocation. The extra red

signal confirms this is not a false positive

result.

METAPHASE FISH

INTERPHASE FISH

Genetic diseases identified using FISH

Prader-Willi SyndromePrader-Willi syndrome is a complex genetic condition that affects many parts of the body. In infancy, this condition is characterized by weak muscle tone (hypotonia), feeding difficulties, poor growth, and delayed development. Beginning in childhood, affected individuals develop an insatiable appetite, which leads to chronic overeating (hyperphagia) and obesity. Some people with Prader-Willi syndrome, particularly those with obesity, also develop type 2 diabetes mellitus (the most common form of diabetes).

Prader-Willi syndrome is caused by the loss of function of genes in a particular region

of chromosome 15.

Angelman SyndromeAngelman syndrome is a complex genetic disorder that primarily affects the nervous system. Characteristic features of this condition include delayed development, intellectual disability, severe speech impairment, and problems with movement and balance.

DiGeorge and velo-cardio-facial Syndromes

It is caused by deletion of small piece of

long arm of chromosome 22 near the

middle at a location designated as

22q11.2

Deletion detected by FISH

Deleted region of

chromosome 22-no flourescnt

signal

intact chromosome 22 giving a fluorescent

signal

• Miller-Dieker Syndrome

• Williams Syndrome de Williams

• Wolf-Hirschhorn Syndrome

• Smith-Magenis Syndrome

• Kallmann Syndrome etc. are the other methods.

Applications of FISH DiagnosticsFISH is often used in clinical studies : If a patient is infected

with a suspected pathogen, bacteria, from the

patient's tissues or fluids, are typically grown on agar

to determine the identity of the pathogen. Many

bacteria, however, even well-known species, do not

grow well under laboratory conditions.

FISH can be used to detect directly the presence of

the suspect on small samples of patient's tissue.

FISH can also be used to compare the genomes of

two biological species, to

deduce evolutionary relationships.

FISH is widely used in the field of microbial ecology,

to identify microorganisms.

Often parents of children with a developmental

disability want to know more about their child's

conditions before choosing to have another

child. These concerns can be addressed by

analysis of the parents' and child's DNA. In

cases where the child's developmental disability

is not understood, the cause of it can potentially

be determined using FISH

and cytogenetic techniques.

In medicine, FISH can be used to form

a diagnosis, to evaluate prognosis, or to

evaluate remission of a disease, such

as cancer.

o FISH is performed on a variety of specimen types such as blood, bone

marrow and paraffin-embedded tissue sections.

o Because of the higher sensitivity, FISH testing can detect mosaicism for

clonal chromosome changes,and is therefore useful in the detection of

minimal residual disease following treatment (e.g., interferon for chronic

myelogenous leukemia [CML]).

o Because the majority of specific deletions are sub-microscopic and can

only be detected by molecular tests, FISH plays a critical role in the

confirmation of microdeletion syndromes.

o FISH is also useful in solid tumors. For example, in determining the

prognostic role of HER-2/neu gene amplification or overexpression in

breast cancer.

Presented by :

Name : ANJALI BAJAJ

Roll No :1754