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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. Church buffer (see table 2.4) was chosen as the hybridisation solution because it produces autoradiographs with little background. 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

Table 2.4. 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 A^^o OD units/ml random hexanucleotides in TE

-Mix solutions A, B, C in the ratio of 2:5:3

Hybridisation buffer:

Church: 7% SDS

0.5 M NaPi, pH 7.2 1 mM EDTA

1% Bovine serum albumin - 1 M NaPi is:

71 g Na2HP04 in 1 litre ddH 20 -pH 7.2 with phosphoric acid

method. Membranes were prewetted in 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. Membranes were prehybridized from 4-16 hours at 65®C to block any physical trapping of the probe.

2.5.1. Addition of Probe

The separated probe (section 2.4.4) 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 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 in Sealey et al (1985) but, in most cases, successful results were obtained by adding 5 |il of lOmg/ml Cot-1 DNA, 50 p,l of 20 x SSC, 25 p.1 of ddH20 and 20 |il of 0.1% (w/v) SDS to 100 |il 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 prehybridization stage, with the addition of 1 0 0 fig/ml of sheared human placental DNA to the hybridisation fluid which acted to block out any repetitive sequences contained in the DNA on the filters.

2.5.2. Washing

Non-specific binding of unincorporated nucleotides and probe DNA occurs in spite of adequate prehybridisation. Washing aims 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. The radioactive hybridisation solution was poured off and the remaining traces removed by swilling the bottle out with 100 mM NaPi/0.1% SDS. 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.

2.5.3. 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 proof cassettes and exposed to XAR-5 autoradiography film (Kodak) with intensifying screens (Kronex Quanta 3, DuPont) at -70®C for 16 hr and the film was then developed. The position of a black band on autoradiography denoted the presence of a sequence 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 reused 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.