Fibra óptica utilizada por Telconet S.A

DNA was electrophoresed on an agarose gel, either 2.5% Nusieve agarose (FMC Biotechnologies) for fi*agment sizes of less than 1 kb, or 0.8% Seakem agarose (FMC Biotechnologies) for fi*agments larger than 1 kb. The buffer used for electrophoresis was 0.5 X E containing 0.5 p g / m l ethidium bromide added to stain the DNA. The conditions for electrophoresis differed depending upon the size and composition o f the

gel. Once the DNA had electrophoresed into the gel, the gel was destained for 1 hour in H2O. If the fragments were o f a large size the gel was depurinated in HCl for

10 minutes, for smaller fragments this step was omitted. The gel was then soaked in 0.4M NaOH for 20 minutes. A nylon filter, Hybond N+ (Amersham), was pre-wetted in H2O and soaked for 5 minutes in 0.4 M NaOH. A wick o f blotting paper (3MM paper, Whatman) was soaked in 0.4 M NaOH. The gel was placed upon this wick, the Hybond filter was placed on top of the gel and covered with 5-10 sheets o f pre-soaked blotting paper, and absorbent tissue. A 1 kg weight was placed on top o f the tissue and this was left overnight at room temperature whilst the DNA transferred to the Hybond filter. Once the DNA had transferred, it was washed twice in 2 x SSC (20 x SSC 3 M sodium chloride, 0.3 M sodium citrate) and stored, wrapped in Saran wrap, at either 4°C for short term storage, or at -70°C for long term storage. To label the DNA, the filter was rinsed in 2 x SSC and placed in a hybridisation tube with hybridisation buffer, either standard hybridisation buffer [4 x SSC, 10 x Denhardfs

{2% (w/v) Polyvinyl pyrolidine, 2% (w/v) Ficoll, 2% (w/v) bovine serum albumin (BSA)}, 0.1% (w/v) sodium pyrophosphate, 5% (w/v) dextran sulphate, 50 pg / ml sonicated salmon sperm DNA], or when labelling with a d(C.A)n-d(G.T)n

heteropolymer (Pharmacia Biotech), using Church buffer (0.5 M disodium hydrogen phosphate, 7% (w/v) SDS, 1 % (w/v) BSA, 1 mM EDTA). The filters were incubated in this buffer for 6 hours at 65°C.

The probe for hybridisation was prepared using one o f two methods, random primer labelling (section 2.6.1) or nick translation (section 2.6.2).

The labelled probe was heat denatured at 95°C for 3 minutes, then placed on ice for a fiirther 2 minutes. The probe was then added to prewarmed hybridisation buffer, (Church buffer was used for the d(C.A)n-d(G.T)n heteropolymer). The filters to be probed were conditioned in prehybridisation buffer for 6 hours at 65°C. When ready to perform the hybridisation reaction the prehybridisation buffer was removed from the filters and 10 ml o f fresh hybridisation buffer containing the denatured probe were added. The D N A bound to the filters was hybridised for 12 hours at 65°C. When using labelled cosmids as probes, or other D N A that contained (or was likely to contain) repeat sequences, the protocol was modified to remove the repeat sequences by incubating the labelled probe with total placental human D N A (Sigma) prior to adding it to the filter (section 2.6.3).

After hybridisation the filters were washed twice for 15 minutes in 2 x SSC 0.1% SDS (w/v), twice in 1 X SSC 0.1% SDS, and tAvice in 0.1 x SSC 0.1% SDS at room

temperature, then twdce in 0.1 x SSC 0.1% SDS at 65°C. The filters were wrapped in Saren wrap and exposed to photographic film.

2.6.1 Random primer labelling.

Labelling was carried out using the Amersham Megaprime kit. 25 ng o f DNA was added to 5 pi of nonomer primer and sufficient H2O added to make the final volume

50 |il. The sample was incubated at 95 °C for 5 minutes to separate the DNA strands and allowed to cool to room temperature to allow the nonomer primers to anneal to the DNA. 5pi o f labelling buffer (10 x concentrated buffer containing Tris/HCl pH 7.5,

P- mercaptoethanol and MgCb) was added, together with 4 pi o f each dNTP,

(omitting the dNTP (s) used to label the DNA), 5 pi o f the labelled dNTP, (^^P dNTP, 50 pCi, 3000 Ci/mmol (Amersham)) and 2pi o f Klenow fragment DNA polymerase. The sample was mixed gently and incubated at 37°C for 10 min. The labelled DNA fragment was purified by centrifugation at 100 0 x g for 6 minutes at room temperature through a pre-prepared Sephadex spin column, which retains free nucleotides in the column matrix.

2.6.2 Labelling by nick translation.

150 ng o f the probe DNA to be labelled was added to 20 pi o f nucleotide labelling buffer (100 pM each ofdATP, dGTP, and dTTP in Tris/HCl pH 7.8,

2-mercaptoethanol, and MgCb, 10 pi ^^P dCTP [50 pCi, 3000 Ci / mmol

(Amersham)], and H2O was added to give a final volume o f 90 pi. 10 pi o f enzyme (DNA polymerase I and DNase I) was added and the sample incubated for two hours at 15°C and the labelled DNA fragment purified by centrifugation using a Sephadex spin column (see above).

2.6.3 Preparing DNA with repeat seq u en ces for Southern blots

For DNA with repeat sequences, the DNA was labelled as described above, and the Southern filters prepared in the same way. When the filters were prehybridised,

sonicated human placental DNA (Sigma) at 200 p g /m l was added. The labelled DNA probe was prepared as described above, and 6 mg o f sonicated human placental DNA added. The sample was heated to 100°C for 5 minutes, cooled to 65 °C and maintained at this temperature for 5 minutes. The DNA probe was then added to a solution containing 400 pi 20 x SSC and 400 pi o f 25 % dextran sulphate (to give a final concentration o f 5 x SSC, 5 % (w/v) dextran sulphate), with sufficient H2O to make a total volume o f 2 ml. This was incubated at 65°C for 6 hours before being added to the hybridisation buffer, which was then added to the filter on which the DNA to be probed was bound.