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Hybridisation reactions are based on the ability of denatured DNA to anneal to a complementary strand present in a nearby environment at a temperature below that of theirs.
The two important features of hybridisation are:
(1) These reactions are specific i.e. the probe will only bind to targets with complementary sequence.
(2) These reactions will occur in presence of large quantities of molecules, similar but not identical to the target.
This means that a probe can find one molecule of target in a mixture of billions of related but non-complementary molecule. Because of these properties it is possible to use hybridisation for molecular search for one DNA molecule in a complex mixture of many similar molecules. Hybridisation is essential because, when a cell is broken to extract DNA, it will be a mixture of the entire cell’s DNA which has thousands of genes and is impossible to study a specific gene.
Hybridisation buffers, usually consist of four components:
(i) Detergents and blocking agents like Denhardt’s,
(ii) Milk powder,
(iii) Heparin and
(iv) Detergents like SDS used to suppress non-specific binding of the probe to the membrane.
Salmon sperms or calf thymus have heterologous DNA that reduce the non-specific binding of probes to non-ho- mologous DNA on the blot. Components like dextran sulphate and polyethylene glycol have large hydrated volumes and increase the viscosity and hence, enhance the rate and extend of hybridisation.
High salt buffers like Saline Sodium citrate (SSC) and SSPE provide high ionic strength necessary for quick hybridisation and form the basis of all hybridisation and washing solutions. Sometimes denaturants like urea and form-amide are also used. These, lower the melting temperature of the hybrid, thus making it possible to carry out hybridisation at lower temperatures.
A. Prehybridisation:
Prehybridisation (prior to hybridisation) is carried out in which potential binding sites for the probe other than homologous DNA are blocked to avoid background hybridisation to membrane. Filters are prehybridised at least for 2 hours in the buffer containing SSC/SSPE and blocking agents like Denhardt’s SDS and non-specific DNA. After hybridisation, unbound labelled DNA is removed from the membrane by washing the filters.
Washing is mainly done to remove non-specific hybrids and thus for reducing the background stringency of washes. This is usually determined by temperature and salt concentration of post- hybridisation washes. Stringency increases with decreasing salt concentration, increasing detergent concentration and temperature.
Requirements:
1. Hybridisation chamber.
2. Microcentrifuge.
3. Waterbath (regulated).
4. Bottle
5. Membrane.
6. Micropipettes.
7. Forceps.
8. Vortex mixer.
9. Orbital shaker.
10. U.V. illuminator and cross linker.
11. Exposure cassettes.
Reagents:
Procedure:
1. Pre wet the blot separately in 2x SSC.
2. Roll the membrane on to the outside and insert into the bottle.
3. Place the bottle on a flat surface and roll it slowly in a direction opposite to that in which the membrane is rolled and if there is any 20x SSC in the bottle, discard it.
4. Add 10-15 ml of prehydration buffer to the bottle, tightly place the lid and place it into the rotisserie with another bottle to counter balance.
5. Place the rotisserie in a previously set oven at 65°C. The bottles should rotate in the same direction as that of the rolling of membrane.
6. Set the speed at 4-6 rpm and prehybridise for at least 2 hours.
B. Hybridisation:
This is done to create a radioactive copy of a double stranded DNA fragment which starts with a restriction fragment of a plasmid containing the gene of interest. For this the plasmid is digested with certain restriction enzymes, and the digest is run on an agarose gel. The plasmid, which usually is less than 20 kb long will give rise to fragments of different lengths.
The desired band can be identified on the gel if the restriction map of the plasmid is known. The band is then cut out of the gel, and DNA is eluted. Since the bands were well separated, the isolated DNA is a pure population of identical double stranded DNA fragments.
The DNA template (restriction fragment) is then labelled by Random Hexamer Labelling. Denature the DNA template by boiling. A mixture of DNA hexamers (six nucleotides of ssDNA) having all possible sequences is added to the denatured template and allowed to base pair. At many sites along each strand of DNA they pair. Add DNA polymerases along with dATP, dGTP, dTTP and radioactive dCTP.
Requirements:
Reagents:
Procedure:
1. Denature the pooled probe fractions in a boiling water-bath for 10 minutes and chill on ice immediately for 5 minutes,
2. Add labelling mixture, Klenow and α32P dCTP. Mix well and incubate for 2 hours at 37°C.
3. Dilute reaction mixture to 450 µl with
T.E and add 45 µl of freshly prepared 2(N) NaOH to denature labelled DNA.
4. Add the labelled probe to the hybridisation solution avoiding contact with the membrane and incubate at 65°C for at least 16 hours (overnight) to allow probe molecules to find their targets.
C. Washing of Membrane:
After formation of hybrids between the probe and the target, any probe which is not stuck to target should be washed and removed. Otherwise the whole filter will become radioactive and specific hybrids will be undetectable.
Requirements:
Reagents:
Prepare wash solutions A and B and preheat at 65°C.
Procedure:
1. Pour out hybridisation solution (it can be stored at 20°C and reused). Boil with hybridisation solution again for 10 minutes.
2. Add low stringency wash A solution into the bottle (about 30 ml). Give two washes each with a duration of 15 minutes by keeping the bottle on a rotating shaker at room temperature. Pour out the solution.
3. Give one wash for 10 minutes with high stringent wash B at room temperature and pour out the solution.
4. Remove the membrane from the bottle and blot excess solution by placing it on Whatman No. 1 filter paper.
5. Check the counts on the blot by hand monitor and expose it to an X-ray film in a cassette using intensifying screen at -40°C.
D. Autoradiography:
The site of hybridisation of radioactive nucleic acid (32P) is visualised by autoradiography. It produces permanent images of the distribution of radioactive nucleic acid on photographic film. The membrane with radioactive nucleic acid is brought in close contact with X-ray film in a cassette. 32P emits high energy particles which pass completely through X-ray film and hit the intensifying screens at the top and bottom of the cassette.
The screens are excited by the radiation and emit photons which are captured by the silver halide crystals in the emulsion on the X- ray film. This causes approximately five fold enhancement in the intensity of the image when the film is exposed at a temperature of -40°C.
Requirements:
X-ray developer (IX)
Dissolve components one by one and make up the volume to 1000 ml.
X-ray film fixer (IX):
Dissolve components one by one and make the volume to. 1000 ml.
X-ray film and cassette.
Procedure:
1. Fix the membrane (in dark room) in a light tight cassette with tape with DNA side of the membrane facing up.
2. Place the sheet of X-ray film and fix it with tape and close the cassette.
3. Expose for an appropriate time at-40°C.
4. Remove the film from storage and fix it in a film holder.
5. Develop the film in a dark room under red safety light as follows:
In x-ray developer: 2 minutes
Rinse with water
X-ray fixer: 4 minutes
Running water: 15 minutes
Air dry film