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Genomics
FISH – STS Mapping
Presented By;
Girish Kumar K
III Msc Biomedical Science
FISH
• FISH stands for Fluorescent in situ
Hybridization.
• FISH is a process that vividly paints
chromosome or a portion of chromosome
with fluorescent molecule.
• It is cytogenic technique that uses
fluorescent probes to bind to the part of the
chromosome.
• It was developed in 1980 and was used to
detect the presence or absence of DNA
sequence.
• The location at which these probes are
bound to the chromosomes are detected by
using fluorescent microscope.
• FISH is used to identify chromosomal
abnormalities, helps in gene mapping,
toxicological studies, analysis of
chromosomal structure aberration and
ploidy determination.
Procedure
• Denature the chromosomes
• Denature the probe
• Hybridization
• Fluorescence staining
• Examine slides or store in the dark
• A probe is constructed, it must be large enough to
hybridize the specific target.
• The probes is tagged with flurophones to the target
with specific antibodies or with biotin. One of the
common tagging method is nick translation.
• A chromosome in interphase or metaphase is taken and
attached to a substrate, usually glass.
• Repetitive DNA sequencing must be stopped by adding
short DNA fragment to the sample.
• The probe is applied to the sample and incubated for
12hours.
• The results are visualized under a fluorescent
microscope.
• Amplification is done if the signal is too weak.
• The signal strength depends on labeling efficiency,
type of probe and type of dye.
• To amplify the signal a compound called
streptavidin.
FISH Procedure
DETECTION
DENATURE OF
PROBE
DENATURE OF
DNA
Interpretation of FISH
Each fluorescently labeled probe that hybridizes to a
cell nucleus in the tissue of interest will appear as a
distinct fluorescent dot.
 Diploid nuclei will have two dots.
 If there is duplication in the region of interest,
the gain will result in more than two dots.
 If there is a loss in the region of interest, one or
zero dot will result.
STS Mapping
• STS stands for Sequence Tagged Site. In STS
mapping the positions of short sequences are
mapped by PCR and/or hybridization analysis of
genome fragments.
• It is short DNA Sequence of 200-500 bp.
• It has only single occurrence in the genome whose
location and base sequence are identified.
• This STS can be detected by using PCR
(Polymerized Chain Reaction).
• It is done by employing specific primers.
• When STS loci contain any polymorphism the
become valuable genetic markers.
• They are used in short gun sequencing.
• STS is used in detecting micro deletion in some
genes.
PCR confirmation of STS markers in the genome
Each STS contains a unique sequence
Mapping Techniques - Fluorescent in situ Hybridization(FISH) and Sequence Tagged Site(STS)

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Mapping Techniques - Fluorescent in situ Hybridization(FISH) and Sequence Tagged Site(STS)

  • 1. Genomics FISH – STS Mapping Presented By; Girish Kumar K III Msc Biomedical Science
  • 2. FISH • FISH stands for Fluorescent in situ Hybridization. • FISH is a process that vividly paints chromosome or a portion of chromosome with fluorescent molecule. • It is cytogenic technique that uses fluorescent probes to bind to the part of the chromosome.
  • 3. • It was developed in 1980 and was used to detect the presence or absence of DNA sequence. • The location at which these probes are bound to the chromosomes are detected by using fluorescent microscope. • FISH is used to identify chromosomal abnormalities, helps in gene mapping, toxicological studies, analysis of chromosomal structure aberration and ploidy determination.
  • 4. Procedure • Denature the chromosomes • Denature the probe • Hybridization • Fluorescence staining • Examine slides or store in the dark
  • 5.
  • 6. • A probe is constructed, it must be large enough to hybridize the specific target. • The probes is tagged with flurophones to the target with specific antibodies or with biotin. One of the common tagging method is nick translation. • A chromosome in interphase or metaphase is taken and attached to a substrate, usually glass. • Repetitive DNA sequencing must be stopped by adding short DNA fragment to the sample. • The probe is applied to the sample and incubated for 12hours.
  • 7. • The results are visualized under a fluorescent microscope. • Amplification is done if the signal is too weak. • The signal strength depends on labeling efficiency, type of probe and type of dye. • To amplify the signal a compound called streptavidin.
  • 9. Interpretation of FISH Each fluorescently labeled probe that hybridizes to a cell nucleus in the tissue of interest will appear as a distinct fluorescent dot.  Diploid nuclei will have two dots.  If there is duplication in the region of interest, the gain will result in more than two dots.  If there is a loss in the region of interest, one or zero dot will result.
  • 10. STS Mapping • STS stands for Sequence Tagged Site. In STS mapping the positions of short sequences are mapped by PCR and/or hybridization analysis of genome fragments. • It is short DNA Sequence of 200-500 bp. • It has only single occurrence in the genome whose location and base sequence are identified.
  • 11. • This STS can be detected by using PCR (Polymerized Chain Reaction). • It is done by employing specific primers. • When STS loci contain any polymorphism the become valuable genetic markers. • They are used in short gun sequencing. • STS is used in detecting micro deletion in some genes.
  • 12.
  • 13. PCR confirmation of STS markers in the genome Each STS contains a unique sequence