Monday, September 24, 2007

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

Image of HYBrite system

Adapted from:

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


MedBankers said...


why is there a need to have a temmperature during the post hydridization washing? from 75 to R.T.P?

BloodBank.MedMic.Haematology said...
This comment has been removed by the author.
BloodBank.MedMic.Haematology said...

hey Yvonne

wanna ask.. is there any abnormalities that cannot be detected by FISH? why if there is..

Doreen(tg 01)

royal physicians said...


May i know what is a "diamond pen"???

And also, i've always heard my collaeague say it's so hard to get the fluorescence signal they wanted. So are u facing similar problems too? =)



first6weeks said...


you mentioned about "Seal the edges with rubber cement". may i know what is the purpose in doing so?

kai lin

first6weeks said...


Just wondering, what is the purpose of FISH and when it is require?


J.A.M.M.Y.S said...

To doreen,

Yes, there are abnormalities which are undetectable by FISH, such as structural chromosomal abnormalities, mosaicism and trisomies other than 13,18,21,X and Y.
This is because FISH is a highly specific technique and it only pick up targets which are requested for.

To Kangting,

the diamond pen is used to label on the glass slide, indicating the spot where the suspension was dropped. We do not face such problem but the main challenge is to judge the signal.

To Kailin,

We seal the edges of the coverslip with rubber cement to prevent any suspension from sipping out of the slides.

To Juexiu,

FISH uses short sequences of
single-stranded DNA (probes) that carry fluorescent tags to
detect chromosomal DNA with a complementary sequence. This test is highly specific and are able to detect microdeletions. It is used in any laboratory, usually molecular laboratory, to detect chromosomal abnormalities.