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The technique of Southern hybridisation involves radioactively labelling a DNA probe, denaturing it to render it single stranded, and applying it to a membrane to which single stranded DNA has been fixed. If a sequence complementary to the DNA probe exists on the membrane, the probe will hybridise to it and be recognised as a black band on X-ray film laid on top of the hybridised membrane (autoradiography). The composition of all the buffers used is shown in table 2.6.

2.5.4.a Oligolabelling

The aim is to generate a single-stranded copy of the probe labelled with a substance that allows detection of the probe once it has hybridised to homologous sequences immobilised on the membrane. Although detection systems based on colour change, or light production exist the most sensitive method remains labelling the probe with radioactivity. Oligolabelling results in exact copies of the single stranded probe being made using random hexanucleotides, non-radioactive deoxyribonucleotides dATP, dGTP and dTTP and radioactive dCTP, and the reaction is catalysed by the Klenow fragment enzyme as described in Feinberg & Vogelstein (1983).

70 ng of probe DNA was made up to a total volume of 21 |il with ddH20, boiled for 3 min to denature the DNA and snap cooled on ice. 3 |il of lOx oligolabelling buffer (OLE) and 1.5 |il of 10 mg/ml bovine serum albumin (BSA) were added followed by 3 |xl radio-phosphorous labelled deoxycytidinetriphosphate (^^p dCTP, >5000 Ci/mmol). 1.5 jil of Klenow DNA polymerase (NBL) was added to the reaction mix which was left at room temperature in a perspex box for 4 - 16 hours. To increase the specificity of the hybridisation, the labelled probe was separated from the un-incorporated ^^p dCTP by elution through a sephadex G-50 column. Sephadex G50 was autoclaved with sufficient TE to ensure full adsorbtion of fluid to the Sephadex beads. A ball of polymer wool was inserted into a 145mm glass Pasteur pipette and Sephadex suspension added until the Pasteur pipette was full. TE drips out of the column leaving compacted Sephadex beads. The column was clamped vertically on a retort stand and a rack containing nine eppendorf tubes positioned below. The 30 p.l of labelled probe mix was added to the column followed by 150 |il aliquots of TE. The first four 'drip through' aliquots were collected in a single eppendorf but, thereafter, each 150 ql aliquot was dripped into a separate tube until all nine tubes had been used. Tube 1 contained 600 ql, tubes 2 to 9 150 jil. Each tube was placed 20 cm from a collimated Geiger Muller monitor (Mini-L. Mini Instruments Ltd., Essex, UK). The eluted probe formed the first 'peak' of radioactivity, normally tubes 4-6, after which the radioactive signal declined until tube 8 and 9 in which the second peak represented un-

The specific activity of the probe was calculated from a 5 p.1 sample using a Bioscan QC 2000 counter (V.A.Howe Co. Ltd., London, UK). The specific activity was usually in the region of 2 x 10^ cpm /|ig of probe DNA but this depended on the quality of the DNA and the age of the radionucleotides used.

Table 2.6

Solutions used in Southern hybridisation:

lOx Oligolabelling buffer:

Solution A: 1.25 M Tris-HCl, pH 8

125 mM Magnesium chloride 225 mM 6-mercaptoethanol 0.5 mM of dGTP, dATP, dTTP Solution B: 2 M Hepes, pH 6 with NaOH

Solution C: 90 OD A^^^ units/ml random hexanucleotides in TE -Mix solutions A, B, C in the ratio of 2:5:3

Hybridisation buffers:

Denhart's: 4x SSC

lOx Denhart's solution 0.1% SDS

0.05 mg/ml boiled, sonicated salmon sperm -lOOx Denhart's solution is :

2% (w/v) bovine serum albumin 2% (w/v) Ficoll

2% (w/v) polyvinylpyrrolidone Church: 7% SDS

0.5 M NaPi, pH 7.2 1 mM EDTA

1% Bovine serum albumin - 1 M NaPi is:

71 g Na2HP0 4 in 11 ddH20 -pH 7.2 with phosphoric acid

2.5.4.b Hybridisation

The aim of the hybridisation technique is to:

• prepare the membrane so as to minimise the non-specific binding of the radioactive probe.

• To add single stranded probe DNA

• To perform the hybridisation reaction in such a way as to optimise the specificity of annealing between probe and membrane bound homologue without decreasing sensitivity. This could be achieved by altering the type of hybridisation fluid used and introducing competitive binding of any repetitive DNA sequences in the probe.

Hybridisation solution needs to contain a wetting agent, a salt concentration which allows annealing of homologous sequences with little non-specific binding and a 'blocking agent' to prevent physical trapping of radioactive probe in the irregularities in the membrane. The most frequently used hybridisation solution was Church buffer, which produced autoradiographs with little background signal. However, some probes produced poor signal intensity with Church buffer and for these the less stringent Denhart's solution was used. Both solutions were used for hybridisation at 65"C. It is important that the membrane is in contact with freely circulating hybridisation fluid at a strictly controlled temperature and I found that placing the membrane in rotating cylindrical glass bottles in a hybridisation oven (Hybaid, UK) to be the most efficient method. Membranes were prewetted in 2 x SSC if Denhart's hybridisation solution was used or 0.5 M NaPi for use with Church buffer. The membrane was placed on a nylon mesh in the appropriate solution and, if more than one membrane was to be hybridised with the same probe, a further nylon mesh was inserted between each membrane to ensure adequate circulation of hybridisation solution. The membranes were rolled up, placed in a pre-warmed glass hybridisation bottle and 20 ml of 2 x SSC or 0.5 ml NaPi at 65°C was added. The hybridisation bottle was then rolled in the direction which caused the membrane to adhere to the sides of the bottle. The solution was poured off, hybridisation solution was added (minimum 15 ml) and the bottle placed in the rôtisserie of the preheated hybridisation oven in such a way that the direction of rotation of the rôtisserie did not cause the membranes to roll up again and lose contact with the hybridisation fluid.

The membranes were 'prehybridised' (to block any physical trapping of probe) for 2 hours if Church buffer was used or for 16 hours in Denhart's was used. Only in the case of Denhart's solution was the hybridisation solution replaced with a fresh aliquot after prehybridisation.

2.5.4.C Addition o f Probe

The separated probe was boiled for 3 min, snap cooled on ice and added to the hybridisation fluid in the cylindrical glass bottle, aiming to achieve a specific activity of 1 X 10^ cpm per ml of hybridisation solution. Hybridisation was for 16 hr at 65°C. However, if the probe was known to contain repetitive elements, their effect could be reduced by competitive binding with Cot-1 DNA (total human DNA enriched for repetitive sequences, Gibco BRL). The exact amount of competition can be calculated as described by Sealey et al (Sealey et al, 1985) but in most cases successful results were obtained by adding 5 pi of lOmg/ml Cot-1 DNA, 50 pi of 20xSSC, 25 pi of ddH2Ü and 20 pi of 0.1% (w/v) SDS to 100 pi of the separated probe, boiling for 3 min and then allowing it to anneal at 65“C for 1 hr. The probe was then added to the hybridisation fluid in the bottle without further boiling. Competition could also be applied at the oligolabelling stage, and the addition of 100 pg/ml of sheared human placental DNA to the hybridisation fluid further reduced the effect of repetitive elements in the probe.

2.5.4.d Washing

Non-specific binding of un-incorporated nucleotides and probe DNA occurred in spite of adequate prehybridisation. Washing aimed to displace the non-specific hybridisation but leave the probe hybridised to homologous sequences on the membrane. The stringency of washing is determined by salt concentration and temperature: the lower the salt concentration and the higher the temperature, the more stringent the wash. Reducing salt concentration is a greater change in stringency than increasing the temperature. The radioactive hybridisation solution was poured off and the remaining traces removed by swilling the bottle out with 2 x SSC 0.1 % SDS for Denhart's, 100 mM NaPi 0.1% SDS for Church buffer. If the washing conditions for the probe were known, the correct washes were performed in the hybridisation bottle, if not, sequential increases in stringency were applied. Deciding whether to increase the stringency of the wash was based on checking the filter with a Geiger Muller monitor (Mini-L, Mini Instruments Ltd., Essex, UK). High generalised activity indicated the need for more stringent washing , or the presence of repetitive sequences in the probe.

2.5.4.C Autoradiography

Once washed, filters were blotted with 3MM Whatman paper to remove surface fluid and sandwiched between pieces of Saran wrap. They were then placed in light

homologous to the probe at the corresponding position on the filter. If the band appeared pale, the exposure time was increased. To allow the membranes to be re-used more quickly than if the radioactivity band was allowed to decay naturally, the radioactive probe was stripped off by immersing the membranes in boiling 1% SDS, 10 mM Tris HCl pH 8 and leaving to cool to room temperature with vigorous agitation.