SPRINT 2 “ACTUALIZAR PRODUCTOS Y ASIGNACIÓN DE

Two probes for the 5a-R I gene were used to screen the genomic DNA library.

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Probe A l : Probe A1 (described above), was radiolabelled using a oligolabelling kit (Pharmacia Biotech) following manufacturer’s instructions. This kit uses Klenow DNA polymerase, random hexamers and labelled dCTP to label DNA. 0.5pg of plasmid DNA was added to 37.5pl of double distilled water, heated to 95°C for 5 minutes to denature and placed on ice. To the DNA lOpl of buffer containing dATP, dGTP, dTTP and random

hexamers, Ip l Klenow polymerase and 2.5pl of dCTP were added and incubated at 37°C for 1 hour. The radiolabelled probes were then separated from excess label using a spin column (Pharmacia Biotech) used according to the manufacturer’s instructions. IpL of the labelled probe was added to 5ml of scintillation fluid and radioactivity determined in a scintillation counter.

Probe A 2: Probe A2 (described above) was 5' end labelled using T4 polynucleotide kinase (PNK, Pharmacia Biotech). To 0.5|Xg of DNA 2|xl of lOX kinase buffer, 5|xl of ô P^^ ATP (50|LiCi) and Ijil of PNK (lOU) were added and incubated at 37°C for 30 minutes. Unincorporated label was removed by passing through a Sephadex G50 column.

Probe A 3: To obtain probe A3 to screen the library two PCR primers (described above) which would amplify 193bp product corresponding to partial exon I of the 5a-reductase I gene was synthesised. These purified primers were used to amplify human colon genomic DNA. 50)il of PCR reaction mixture contained l|xl of human colon genomic DNA, lOpmol of each primer, 100|iM dNTP, 5|xl of lOX PCR buffer and 5U of Taq polymerase. After a hot start of 94°C for 5 minutes, PCR was carried out at 94°C for 45 seconds, 60°C for 45 seconds, 72°C for 45 seconds for 30 cycles followed by a final extension step of 72°C for 10 minutes. The products were run on a 4.5% polyacrylamide gel and a band excised whose mobility corresponded to the expected 193bp product. It was processed as described in isolation and reamplification of PCR products (described below). The fragment was sequenced (described below) using the original PCR primers and when the sequence of the fragment was confirmed it was radiolabelled as described for probe A2.

Probes fo r 5 a-R II gene

Two probes for the 5a-R U gene were used to screen the genomic DNA library.

Probe B l : Probe B1 (described above) was random labelled using a oligolabelling kit as described for probe A l above.

Probe B2. To obtain probe B2 to screen the library, two PCR primers (described above) which would amplify a 214bp product corresponding to partial exon I of the 5a-reductase II

gene were synthesised. These purified primers were used to amplify human colon genomic DNA. 50|il of PCR reaction mixture contained l|il of human colon genomic DNA, lOpmol of each primer, lOOpM dNTP, 5|xl of lOX PCR buffer and 5U of Taq polymerase. After a hot start of 94°C for 5 minutes, PCR was carried out at 94°C for 45 seconds, 55°C for 45 seconds, 72°C for 1 minute for 30 cycles followed by a final extension step of 72°C for 10 minutes. The products were run on a 4.5% polyacrylamide gel and a band excised whose mobility corresponded to the expected 214bp product. It was processed as described in isolation and reamplification of PCR products (described below). The fragment was sequenced (as described below) using the original PCR primers and when the sequence of the fragment was confirmed it was radiolabelled as described for probe A2.

Isolation, reamplification and sequencing o f PCR products excised from gels

The excised gel containing the fragment of interest was squashed using a plastic coated pestle and mixed with lOOpl TE and 1ml of water and shaken vigorously for 1 hour on an Eppendorf shaker. The eppendorf was centrifuged (at 13,000 rpm) for 30 minutes to pellet the gel. The supernatant containing the DNA was carefully removed with a pipette and placed in another tube. The 1.5ml tube was filled with butanol, mixed and centrifuged for 5 minutes. The top layer was removed, filled with butanol, mixed and centrifuged as above to concentrate the DNA solution. This procedure was repeated until ~0.5ml of DNA suspension was left. The DNA was precipitated with 1/10 volume of 3M sodium acetate, pH 5.2 and 2 volumes of 100% ethanol at room temperature (RT) for 20 minutes and centrifuged for 30 minutes to pellet the DNA. The supernatant was carefully removed with a pipette and the pellet air dried for 15 minutes. The pellet was re-dissolved in 20pl of water. Ijil of this DNA was then used to reamplify the fragment using the appropriate PCR conditions. To check the amplified product, 10|Xl was run on 4.5% polyacrylamide gel. To determine whether the reamplified product contained the sequence of interest, it was sequenced.

Preparation o f PCR products fo r sequencing

To sequence PCR products excess dNTP was removed from the PCR mixture as described below. 35|il of reamplified product was mixed with an equal volume of PEG mix (52.4g polyethylene glycol 8000, 40ml 3M sodium acetate pH 5.2, 1.32 ml IM MgCl2 made up to

200ml with water) in an eppendorf tube, vortexed and allowed to stand at RT for 10 minutes. Then the tube was centrifuged for 10 minutes and the supernatant carefully removed with a pipette. To the tube 100 |Lil of 100% ethanol was added and centrifuged for 10 minutes. The supernatant was removed with a pipette and the pellet dried for 15 minutes. The pellet was dissolved in 30|xl water. The DNA was then sequenced on an automated DNA sequencer.