Tag: FISH

Molecular Cytogenetic Technique- (Fish)

MOLECULAR CYTOGENETIC TECHNIQUE- (FISH)


  • FISH is a cytogenetic technique that can be used to detect the presence or absence ofspecific DNA sequences (specific gene locus).
  • fluorescent probes bind to only those parts of the chromosome with which they show a high degree of sequence similarity.like lncRNA, mRNA, or miRNA of interest
  • FISH  can  be used for  rapid  identification  of chromosome during  interphase.
  • FISH can be used in metaphase cells to detect specific microdeletions beyond the resolution of routine qtogenetics or identify extra material of unknown origin.
  • it determines if a chromosome has a simple deletion or is involved in a subtle or complex rearrangement. In addition, metaphase FISH can detect some of the specific chromosome rearrangements seen in certain cancers.
  • This technique can be used on formalin-fixed paraffin embedded (FFPE) tissue, frozen tissues, fresh tissues, cells and circulating tumor cells.

Methodology:

  • (FISH) analysis is performed by denaturing the double-stranded DNA in the fixed chromosomes on a microscope slide.
  • Two fluorescently labeled DNA probes are used in combination to analyze each location.
  • The first probe serves as a control and hybridizes with DNA on the target chromosome, The second probe hybridizes to a target location on the individual’s DNA sequence.
  • When a deletion is present, the second probe will not hybridize and no fluorescence will be seen.
  • A duplication will result in two fluorescent spots with the test probe.

Advantages of FISH

  • FISH permits determination of the number and location of specific DNA sequences in human cells.
  • FISH can be performed on metaphase chromosomes,as with G-banding, but can also be performed on cells not actively progressing through mitosis
  • FISH performed on nondividing cells is referred to as interphase or nuclear FISH.

Disadvantages of FISH

  • FISH requires a preselection of an informative molecular probe prior to analysis, So a prior knowledge of the anomaly is needed.

Uses  of  FISH:

  • Detection of numeric abnormalities of chromosomes (aneuploidy). The demonstration of subtle microdeletions
  • Detection of complex translocations not detectable by routine karyotyping
  • For analysis of gene amplification e.g. HER2/NEU in breast cancer or N-MYC amplification in neuroblastomas
  • For mapping newly isolated genesa of interest to their chromosomal loci

Chromosome  Painting:

  • Is an extension of FISH, Probes are prepared for entire chromosomes.
  • The number of chromosomes that can be detected simultaneouslyby chromosome painting is limited by the availability of fluorescent dyes.

Exam Important

  • FISH  can  be used for  rapid  identification  of chromosome during  interphase.
  • FISH can be used in metaphase cells to detect specific microdeletions beyond the resolution of routine qtogenetics or identify extra material of unknown origin.
  • it determines if a chromosome has a simple deletion or is involved in a subtle or complex rearrangement. In addition, metaphase FISH can detect some of the specific chromosome rearrangements seen in certain cancers.
  • It helps in Detection of numeric abnormalities of chromosomes (aneuploidy). The demonstration of subtle microdeletions

 

Don’t Forget to Solve all the previous Year Question asked on MOLECULAR CYTOGENETIC TECHNIQUE- (FISH)

Module Below Start Quiz

Molecular Cytogenetic Technique- (Fish)

MOLECULAR CYTOGENETIC TECHNIQUE- (FISH)

Q. 1 Which of these is not a method for introducing genes into a cell?

 A

Transfection

 B

Electroporation

 C

FISH

 D

Recombination

Q. 1

Which of these is not a method for introducing genes into a cell?

 A

Transfection

 B

Electroporation

 C

FISH

 D

Recombination

Ans. C

Explanation:

Methods for introducing genes into a cell include, transfection (chemical based), electroporation (physical), infection (viral mediated) and recombination techniques.

 
Fluorescence in situ hybridization (FISH) is a cytogenetic technique that uses fluorescence-tagged probes to detect specific DNA sequences in tissue samples. It is not used for introducing genes into the cells.
 
Methods for genetic manipulation:
 
Transfection is the most commonly used method for introducing transgenes into human embryonic stem (hES) cells. This system is based on the use of carrier molecules that bind to foreign nucleic acids and introduce them into the cells through the plasma membrane.
 
Electroporation is a method that employs the administration of short electrical impulses that create transient pores in the cell membrane, allowing foreign DNA to enter into the cells. 
 
Infection is gene manipulation by viral infection which can produce a very high percentage of modified cells. Advantage over other systems are, high efficiency of DNA transfer (almost 100% efficiency) and single copy integrations.
Recombination occurs by physical exchange of DNA. An endogenous gene can be replaced by a transfected gene using homologous recombination. Recombination is the result of crossing-over that occurs at chiasmata and involves two of the four chromatids.
 
Ref: Human Embryonic Stem Cell Protocols By Kursad Turksen, 2006, Page 225

Q. 2 Rapid method of chromosome identification in intersex is:

 A

FISH

 B

PCR

 C

SSCP

 D

Karyotyping

Q. 2

Rapid method of chromosome identification in intersex is:

 A

FISH

 B

PCR

 C

SSCP

 D

Karyotyping

Ans. A

Explanation:

A i.e. FISH
FISH, chromosome painting and spectral karyotyping (SKY) are rapid methods of chromosome identificationQ.


Q. 3

Rapid method of chromosome identification in interphase is –

 A

FISH

 B

PCR

 C

SSCP

 D

Karyotyping

Q. 3

Rapid method of chromosome identification in interphase is –

 A

FISH

 B

PCR

 C

SSCP

 D

Karyotyping

Ans. A

Explanation:

 
FISH is a cytogenetic technique that can be used to detect the presence or absence of specific DNA sequences (specific gene locus) on chromosomes.
It uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity.
Fluorescence microscopy can then be used to find out where the fluorescent probe bound to the chromosome.
FISH can be used for rapid identification of chromosome during interphase.
FISH can be used in metaphase cells to detect specific microdeletions beyond the resolution of routine cytogenetics or identify extra material of unknown origin.
It can also help in cases where it is difficult to determine from routine cytogenetics if a chromo­some has a simple deletion or is involved in a subtle or complex rearrangement.
In addition, metaphase FISH can detect some of the specific chromosome rearrangements seen in certain cancers.

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