RETROACTIVIDAD Y ULTRACTIVIDAD DE LA LEY QUE REGULA

Single strand conformation polymorphism (SSCP) analysis (Orita et aL, 1989; Hayashi, 1991) was used to detect sequence changes that alter the m igration of DNA through a non-denaturing polyacrylamide gel.

2.2.6.1 Radioactive SSCP detection of unknown sequence changes

2.2.6.1.1 Preparation of samples for SSCP analysis

Genomic DNA was amplified by the PCR (2.2.4), in the presence of radiolabelled nucleotides to allow detection by autoradiography. The PCR was carried out in a 50pl reaction volume, using 40pmol of each primer (table 6), w ith substitution of the unlabeUed 0.2m M dCTP w ith IpC i of [a-32p]dCTP (O .lpl of [a-szpjdCTP, 3000Ci/m m ol, ICN) and 0.02m M of unlabelled dCTP.

The PCR-products were diluted by 4-8 fold In a solution of 0.1% (w /v) SDS and lOmM EDTA. For each diluted sample a volume of 4|j1 was mixed w ith an equal volume of formamlde solution (95% (v/v) deionised formamlde; 0.05% (w /v) bromophenol blue; 0.05% (w /v) xylene cyanol; and 20mM EDTA). This m ixture was denatured for 2-3 minutes at 94°C before placing on Ice, prior to electrophoresis.

2.2.6.1.2 Analysis of DNA by non-denaturing polyacrylamide gel electrophoresis

A non-denaturing polyacrylamide gel m ixture was prepared using a stock solution of liquid aciylamide which contained 30% (w /v) aciylamide and 0.8% (w/v) methylene bisaciylamide In dHzO (ProtoGel™). The stock solution was diluted five fold to contain 6% (w /v) aciylamide, 5% (v/v) glycerol and Ix TBE buffer. In distilled water. This m ixture could be stored at 4°C In the dark for several weeks.

Electrophoresis was carried out In a model S2 sequencing gel system (GibcoBRL), which holds a gel 30cm wide x 40cm long and 0.4mm thick. First, the glass plates were washed w ith dilute detergent (Micro, International Products Corp.) and rinsed w ith tap water before washing w ith 70% (v/v) IMS to dry the plates. The smaller of the two plates was then slllconlsed. In a fume hood, by covering one side w ith either Repelcote (BDH) or Slgmacote (Sigma) solution and allowing It to dry for about 10 m inutes. The plates, separated by 0.4mm spacers arranged at the sides and one at the bottom were clamped together w ith bulldog clips to form the gel mould.

Polymerisation of 70m l of polyacrylamide gel m ixture was Induced by adding 0.5m l of fresh 10% (w /v) ammonium persulphate In dH2 0, to promote free radical

form ation, and 60pl of TEMED to catalyse the cross-Unklng. Care was taken not to shake the gel m ixture too vigorously before pouring, since oxygen Inhibits polymerisation. The gel was poured Im m ediate^ and combs w ith 1cm width wells were Inserted at the top. It was allowed to set for at least 1-2 hours before use.

Denatured DNA samples (6-8pl), section 2.2.6.1.1, were loaded onto the gel and electrophoresed overnight at 320-400V, at room temperature. For PCR products of less than 250 bases (exons 2 and 4) the gel was run u n til the xylene cyanol had migrated about 20cm down the gel, while larger fragments of up to 450 bases (exons 1, 3, 5, 6 and 7) were run un til the xylene cyanol was about 30cm down the gel.

2.2.6.1.3 Detection of radiolabelled DNA by autoradiography

Pofyaciylamide gels were placed on 3MM Whatman paper, covered in cling film and dried under vacuum at 80°C for 0.5-2 hours on an ATTA gel diyer (model AEÎ3700). The cling film was removed from the dried gel. The gel was placed in a light-proof cassette and exposed to X-ray fUm, Kodak X-OMAT AR-5, for a suitable length of tim e (1-7 days), at -70°C. Intensifying screens were not included. The film was processed using a Fuji Photo Film Ltd. developer.

2.2.6 2 Detection of polymorphisms by non-radioactive SSCP

PCR am plification (2.2.4) was used to produce DNA for exons 1 and 3, In a 50pl reaction volume w ith 40pmol of each prim er (table 6).

Pofyacrylamide gels, 16cm wide x 20cm long and 1mm thick, were prepared for electrophoresis using the Midigel Protean II apparatus (BioRad). A vertical gel mould was formed by two glass plates which were held apart by 2 side-spacers and w ith a rubber seal at the gel base. The glass plates were washed twice w ith dilute detergent (Micro) and then w ith 70% (v/v) IMS to diy the plates, before use.

The non-denaturing pofyacrylamide gel m ixture was prepared as described (2.2.6.1.2) but the concentrations of glycerol and pofyacrylamide were varied over the ranges 0-5% (v/v) and 6-18% (w /v), respectively, to determine the optimum concentrations and to maximise the differences in m igration of the variant DNA. The gel m ixture was pofymerised by adding a x l/1 0 0 volume of 10% (w /v) APS and x l/1 0 0 0 volume of TEMED, poured into the gel mould and allowed to set for at least 30 minutes.

Each PCR sample was mixed 3:1 w ith loading buffer, containing 15% (w /v) sucrose, 0.05% (w /v) xylene cyanol in dH20. The sample was denaturing for 3 minutes at 94°C and electrophoresis was in Ix TBE buflfer, overnight at 15°C, w ith water- cooling. Exon 1 products were separated at lOOV so that the xylene cyanol dye was at the bottom of the gel and exon 3 products at 200V. A DNA marker, Ikb ladder, was also run for comparison. The DNA was visualised by immersion of the gels in 0 .5 -lp g /m l ethidium bromide solution for 15 minutes and subsequently viewed under a UV Ught.

Ebcon 1 polymorphisms were detected on a 6% (w/v) pofyacrylamide gel containing