Florescence in situ hybridization

Florescence in situ hybridization (FISH), is a technique involving the separation of double stranded DNA by heat and hybridization of the target DNA sequence to the respective probes with a complementary sequence deirectly-labeled with a florochrome. The metaphases to which the probes anneal to are visualized as florescent signals of specific wavelengths under fluorescence microscopy. The number of signals observed in the metaphase spreads/interphase cells would indicate the copy number of the gene/gene loci concerned. This technique allows the identification of microdeleted/duplicated genes, aneuploidy screening (detection of extra chromosomes of chromosomes 13, 18, 21 X and Y), translocated genes in haematological malignancies and gene amplification from cases with cancer of the breast.

I would be explaining on identifying deleted/duplicated genes and loci that are related with microdeletion syndromes in congential disorders such as Prader-Willi/Angelman, Williams and Di-George using FISH techniques.

Samples – cultures of PHA(mitogen)-stimulated lymphocytes cells, amniotic fluids and chorionic villi.

Slide Preparation

1. Etch out the area on the underside of the glass slide with a diamond pen that contains the highest number of metaphase spreads. Slides can be kept in an airtight container at -20°C or 4°C if they are not used immediately.

2. Dehydrate the slide through an ethanol series (70%, 85% and 100% respectively) for 1 minutes each at room temperature. Blow-dry the slide with a hair dryer.

Probe Preparation and hybridization
The following steps must be carried out in reduced lighting as the probes are light-sensitive.

1. Remove the probe vial from the freezer and thaw at room temperature. Centrifuge the vial.

2. Add probe mixture onto the etched area of the slide and cover with a circular glass coverslip.

3. Seal the edges with rubber cement and place the slide into a light proof box.

4. Switch on the 'HYBrite' system and set the desired program (denaturation and hybridization steps).

5. Moisten the toweling paper in the troughs with water. Place the slide onto the hybridization surface, coverslip facing upwards.

6. Ensure that the target area is in contact with the hybridization surfaces and also that all the remaining slide positions are filled with blank slides to even out the temperature.

7. Close the 'HYBrite' lid and commence the program.

Post Hybridization Wash
Carry out the following steps in reduced lighting.

1. Switch on water bath. Set the temperature to 75° C. Prepare two coplin jars for Wash A and Wash B or two plastic slide chambers if only 1 or 2 slides are required for washing.

2. Place the coplin jar/slide chamber containing Wash A in to the water bath when it is just switched on.

3. Leave the coplin jar/slide chamber containing Wash B at room temperature.

4. When the water bath reaches its set temperature, remove the slide from the 'HYBrite' system. Peel off the rubber cement from the coverslip with a pair of forceps. Remove the coverslip.

5. Place the slide in the Wash A and agitate the slide by jiggling briefly for about 3 seconds. Wash for exactly 2 minutes.

6. Remove the slide and transfer to Wash B. Agitate the slide by jiggling briefly for about 3 minutes. Wash for 1 minute.

7. Remove the slide and air or blow dry completely in the dark at room temperature. Apply 10m l of DAPI counterstain (Vectashield mounting medium) and mount a glass coverslip over the hybridize area. Seal with nail vanish.

Image of Digeorge Syndrome

Adapted from :
http://www.pathology.washington.edu/galleries/Cytogallery/main.php?file=intro

Image of HYBrite system

Adapted from:

http://www.ebiotrade.com/buyf/productsf/vysis/img189_1.jpg

Reference:

American College of Medical Genetics Laboratory (1999). Standard guidelines for clinical genetics laboratories, 2nd Ed. Bethesda, MD: ACMG.

By:

Yvonne Lau

0503149G

TG01