Fluorescent in situ hybridization

2.  Fluorescence insitu hybridization (FISH) is a
combination of molecular and cytogenetic
technique that uses flourescent molecules to
“paint” genes or chromosomes.
 Fluorescence insitu hybridization involves the use
of short sequences of single-stranded DNA called
as PROBES which are labeled with fluorescent
tags, to hybridize, or bind, to the complementary
DNA to see the location of those sequences of
DNA under the fluorescent microscope.

4.  FISH has become important adjunct to routine
karyotyping analysis.
 Major limitation of karyotyping is that it is
applicable only to the cells that are dividing or can
be induced to divide invitro.
 This problem can be overcome by FISH which
uses DNA probes that recognize chromosome
specific sequences . These probes are labelled
with flourescent dyes and applied to interphase
nuclei i.e. non dividing cell as well

5. Thus FISH can be used in dividing as well
as in non diving cells means in
METAPHASE CHROMOSOME AND
INTERPHASE NUCLEI.

8.  Make a probe complementary to the
known sequence. While making the probe,
label it with a fluorescent marker, e.g.
fluorescein, by incorporating nucleotides
that have the marker attached to them.
 Put the target chromosomes on a
microscope slide and denature them.

9. Denature the probe and add it to the
microscope slide, allowing the probe to
hybridize its complementary site.
Wash off the excess probe and observe
the chromosomes under a fluorescent
microscope. The probe will show as one or
more fluorescent signals in the
microscope, depending on how many sites
it can hybridize to

12.  By using the DNA probes that are specific for
defined regions of the chromosome, FISH
can be used to demonstrate subtle
microdeletions , complex translocations, and
telomere alterations that are not readily
detectable by routine karyotyping .
 Thus FISH can be used in gene mapping,
identification of chromosomal abnormalities
and identification of cultured/uncultured
microorganisms in environment.

13.  The abilities to detect specific molecular
identities was first demonstrated using
antigen-antibody interactions.
 In 1977, the first antibody dependent
fluorescent detection of nucleic acid was
achieved.
 The first application of fluorescent in situ
detection came in 1980, when RNA was
directly labeled on the 3 end with fluorophore.

14.  In previous studies the probe was labelled
with a radioactive compound [usually tritium]
such that after 5-10 days of exposure to x-ray
film, a pattern of silver grains could be
detected identifying the chromosomal location
of probe .
1. Longer exposure are often required to
produce the measurable singles in the
radiography while FISH is quick and easier to
perform

15. 2. Radioactive probes are unstable, costly
and hazardous.
3.Sensitivity and Specificity is higher with
FISH technique.

16. Three basic types of probes are described:
1.CHROMOSOME PAINTING PROBES:
 Are actually a cocktail of many unique
DNA fragments from along the entire
length of a chromosome such that after
hybridization, whole chromosome
flouresces.

17. 2. REPEAT SEQUENCE PROBES:
 Are isolated from telomere or centromere
regions.
 Centromere probes are usually used in
chromosome enumeration i.e. to detect gain
or loss of specific chromosomes.
 Telomere probes recognizes six base repeat
present at the ends of chromosome and will
confirm the presence or absence of telomeric
regions.

18. 3.UNIQUE SEQUENCE PROBE:
 Also called as Gene or locus probe.
 Usually isolated from cloned DNA of a
disease causing gene or a fragment of DNA
of known location associated with particular
gene.
 This type of probe is used to identify the
presence or absence of gene , gene region or
chromosomal rearrangement of interest.

19.  Subtelomere probes - is a subset of unique sequence
probe.
 Recent studies revealed that there unique sequence
just proximal to the telomere of each chromosome arm
that can be used for specific identification of each arm.
 The Subtelomere probes that have been generated
have become very valuable in characterizing the
cryptic rearrangements by determining if all
subtelomeres are present and located on the correct
chromosome arm , or not, if rearrangement has
occurred.

25. 1.Slide preparation.
2.Hybridization of target chromosome with
probe.
3. Detection under flourescent microscope.

26. 1.Start with chromosome preparation from any cell type.
2.Incubate with 200 ul of DNAase.
3.Wash the slide in 2x conc.of SSC.
4.Rinse the slide in delonised water.
5.Incubate with 200ul of pepsin for 10 min.
6.Rinse the slide in delonised water.
7.wash the slide in 2x conc. Of SSC.
8.Stablize the slide in paraformaldehyde for 10 min.
9.Again wash the slide in 2x conc. of SSC.
10.Dehydrate the slide in ethanol series of
70%,80%,95%.
11.Air dry the slide.

27. 1.Place 30µl of hybridization solution on
each slide and cover with a plastic cover
slip.
2.Denature the slide at 65-70˚C for 5
minutes.
3.Gradually decrease the temperature to
37˚C
4.Hybridize at 37˚C overnight in humidity
chamber.

28. 1.Wash slides in 2x SSC to remove coverslip.
2.Wash the slide in wash buffer.
3.Cool slides to room temprature.
4.Block in a blocking buffer for 20-30 min.
5.Incubate with 50µl of antidody detection
compound for 30-60 min.
6.Counterstain with DAPI solution for 10 min.
7.Rinse briefly and mount in antifade mounting
medium.

30. Prenatal Diagnosis:
o Trisomy (21/18/13/12/9/8)
o Turner Syndrome
o Trisomy X
o XYY Syndrome
o DiGeorge Syndrome
o Triploidy

31. Cancer Diagnosis and Prognosis:
o Bladder Cancer
o Breast Cancer
o Cervical Cancer
o Chronic Lymphocytic Leukemia
o Chronic Granulocytic Leukemia
o Multiple Myeloma

40. 1. The common problem with this technique is
that the lack of signal can occur due to
technical failure of hybridization.
 To eliminate this as a source of error a
minimum of 20 cells must be evaluated and
all the cells must agree in signal count.
2. Selection of specific probe or the probes
that will help to answer a clinical question.