FISH-Fluorescence+in+situ+Hybridization

FISH-Fluorescence in situ Hybridization B. Ly



FISH is a form fluorescent staining. This method helps to differentiate between differing cells of sequences of DNA or RNA. It is useful when trying to determine which genes are located where.

First, a section of DNA is selected to be analyzed. The section is treated with restriction enzymes that cleave the section at the appropriate places. PCR is needed in order to ensure that there is a sufficient amount of genetic material present in order to perform the hybridization. A sample is needed to cover a slide and therefore you need enough to have the whole slide covered. PCR is used when there are small minute amounts of DNA. PCR is also the quickest way of going about this.

Once a sufficient amount of genetic material is present, it is fixed onto a slide. This is done using chemicals that promote adhesion of the molecules to the slide. A slide is prepared by fixing the DNA sample to it. A fixative compound is added to ensure that the sample will securely be on the slide. This is impertinent because the sample needs to stay on the slide as there will be subsequent washes to ensure that the sample fluoresces correctly. Most likely the fixative will be some sort of methanol, acetic acid combination. After the sample has been fixed onto the slide, it is then treated with a temperature gradient. This helps to slowly spread out the sequences of DNA.

A probe with the same sequences as the DNA adhered to the slide is treated with fluorescent dyes. Both the slide and probe are then treated with a chemical that denatures the sequences. This breaks apart the bonds between the double stranded DNA/RNA. Certain probes can be treated with different hued fluorescent dyes allowing more than one type of DNA to be examined at the same time. The samples are left there for about 30 minutes where the sequences would comeback together. Hopefully taking in some of the DNA with the fluorescent material. The fixed DNA on the slide would then attach itself to the parts of the probe. The sample would then take in the fluorescent material of the probe.

The locations of the specific sequences of genetic material can be found by using fluorescent microscopy.