DNA Extraction from Solid Tumours

DNA was prepared using a standard phenol chloroform extraction method (Maniatis et al., 1982). Frozen tissue was placed in a petri dish and the tissue was minced as small as possible using a pair of clean scalpels. This was then transferred to a universal tube containing 10ml STB buffer (appendix 5.1) with 500|ll 10% SDS to which was added 80jll Proteinase K (25mg/ml). The mixture was incubated overnight at 37°C. When digestion was complete the mixture was extracted twice for 10 minutes with phenol and once with chloroform each time taking the aqueous (top) layer after separating the phases by spinning at 2500rpm in a bench-top centrifuge. To the final aqueous phase, 2 volumes ice-cold absolute ethanol were added and the mixture incubated for approximately 30 minutes at -20°C to precipitate the DNA. The DNA was spooled out and redissolved in an appropriate volume of TE buffer. The

concentration of the DNA was established by measuring the optical density at 260nm. For smaller pieces of tissue the DNA preparation was scaled down to a volume of 500 pi and carried out in a microcentrifuge tube.

2.2.3.1 Amplification of DNA by Polymerase Chain Reaction

For this method (Saiki et al., 1988), it was essential that Güson tips and microcentrifuge tubes were prepared, autoclaved and handled wearing gloves so that any extraneous DNA contamination was minimised. All reactions were carried out using an automated DNA thermal cycler (Hybaid). 500ng-lpg genomic DNA was used as the template for polymerase chain reaction (PGR) amplification. The reaction mixture consisted of the DNA template, 50pmoles of each oligonucleotide primer (Oswel), 1-2 units of thermostable Thermus aquaticus {Taq) polymerase (HT Biotechnology Ltd.), 200pM each dNTP (Pharmacia) and lOX PGR buffer (appendix 5.4.7). A standard reaction volume of 25pi was carried out with distilled water and overlaid with 50pl paraffin to prevent evaporation during the PGR. A negative control containing no DNA template was set up alongside the other samples to check for possible contamination by extraneous DNA in the PGR reagents. The thermocycler was programmed with the specific conditions for the amplification of a particular pair of primers. The first stage of the reaction was an initial dénaturation of DNA template at 94°G for 5 minutes followed by 35 cycles of PGR amplification. Each cycle consisted of three steps: dénaturation of the double stranded DNA template at 94°G for 30 seconds, an annealing time and temperature specific for each primer set to anneal to their complementary sequences in the DNA template, and extension of the DNA strands at 72°G for 45 seconds. At the end of the 35 cycles, a period of 10 minutes at 72°G was allowed for the final extension of the DNA strands.

2.2.3.2 Amplification of YAC DNA by A/n-FCR

Human specific Alu primers were used to amplify only the human DNA from the YAGs. lOOng of YAG DNA was used as the template for PGR amplification. The

reaction mixture (appendix 5.4.8) consisted of lOX PCR buffer, 2mM of each dNTP,

Taq polymerase, lOOng YAC DNA, oligonucleotide primers and water. The standard reaction volume of 50pl was carried out. The reaction mixture was denatured at 96°C for 5 minutes, followed by 30 cycles of amplification, each cycle consisted of dénaturation at 96°C for 1 minute, annealing at 40°C for 30 seconds and extension at 72°C for 6 minutes. The final extension was at 72°C for 10 minutes. The concentration of amplified YAC DNA was measured using the fluorometer.

2.2.3.3 Resolution of DNA Amplification by Agarose Gel Electrophoresis

DNA fi-agments were resolved by electrophoresis on 2% agarose gel containing 1 |ll ethidium bromide (lOmg/ml). Upon completion of the DNA amplification, the paraffin was carefully removed and a 8|Xl aliquot of each reaction was run on a minigel in TBE buffer (appendix 5.1) at 50V for 25-40 minutes. Each sample was mixed with l(ll of loading buffer (appendix 5.1) prior to loading on the gel. The amplified DNA was visualised by ultraviolet transillumination.

2.2.3.4 PCR Detection of Restriction Fragment Length Polymorphisms

The amplified DNA was digested with restriction endonuclease (Gibco BRL) in the appropriate enzyme buffer according to the manufacturer's specification. The standard reaction volume was 15|ll. The digest reaction consisted of 1.5|ll restriction enzyme, 1.5pl buffer, 5-7|il amplified DNA and distilled water. The digest mixture was incubated at 37°C or 65°C for 4-12 hours. To check the completion of genomic DNA digestion, the 15|ll digest reaction was run on 2% agarose gel containing Ijll ethidium bromide (lOmg/ml) in TBE buffer at 50V for 30-45 minutes. A 1Kb ladder (Gibco BRL) was used as a reference size markers and electrophoresed alongside the DNA samples on each gel. The DNA fragments were visualised by ultraviolet transiUumination.

2.2.3.5 PCR Detection of Single Strand Conformation Polymorphisms

For the detection of single strand conformation polymorphisms (SSCP), l|ll DNA amplified by oligonucleotide primers was added to 1.5|ll formamide loading buffer and denatured for 5 minutes at 95°C. The separation and staining procedures were carried out using the Phastsystem^^ (Pharmacia). The Phastsystem was programmed for selecting a separation method to run 1 or 2 gels. A Phastgel sample applicator was used to load the samples onto the 20% polyacrylamide gel at the separation compartment. The duration of each method step and the time for sample application was measured in volthours. The gel was removed after a total run of 30- 45 minutes and placed in the development chamber. The development method was selected for silver staining with the appropriate solutions (appendix 5.4.9) made for developing, fixing and staining the gels. Upon completion, between 30-90 minutes, the gel was removed and air-dried.