mapping techniques - fluorescent in situ hybridization(fish) and sequence tagged site(sts)

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 FISHEach 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

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