2.2.2(a). Preparation of DNA for pulsed field gel electrophoresis.
High molecular weight genomic DNA suitable for PFGE was prepared from fresh human blood, lymphoblastoid cell lines, fibroblasts or cancer cell lines according to the methodology described by Schwartz and Cantour (1984). 20 ml of blood was incubated on ice for 30 minutes followed by the addition of four volumes of chilled
AKE lysis buffer (appendix 2A.4.) and a further 60 minute incubation on ice. Lymphocytes enriched during this lysis were centrifuged for 10 minutes at 6(XX)g and 4°C, and the supernatant discarded. The pellet of cells obtained was subsequently resuspended in a small volume of chilled 1 x phosphate buffered saline (PBS) and, following addition of a further 20ml of chilled 1 x PBS, centrifuged as before. This process of centrifugation, resuspension and washing with 1 x PBS was repeated another three to four times. Lymphoblastoid cell lines grown to a cell density of 1 x 10^ cells per ml in RPMI medium (2A.3.) were similarly washed with 1 x PBS. For surface attached fibroblasts and cancer cell lines, cells cultured in two 150ml tissue culture flasks were treated with 0.025% ^ /v trypsin in versene (2A.3.) at 37“C for 10 minutes to remove from the base of the flask. The cell suspension was removed to 20ml centrifuge tubes with the addition of 3 volumes of 1 x PBS followed by successive steps of centrifugation and washing with 1 x PBS as described for blood lymphocytes and lymphoblastoid cell lines.
Cells were counted using a calibrated haemocytometer and adjusted to a concentration of 1-1.5 X 10^ cells/ml. This suspension was in turn mixed with an equal volume of molten 1% ^ /v low melting point (LMP) agarose (Sigma) and aliquoted in l(K)|il volumes into appropriate moulds (Pharmacia LKB). Once set, the blocks were incubaed at 50°C in ESP lysis buffer (2A..4.) for 48hrs with buffer renewal after 24hrs. The blocks were then washed in sterile 1 x TE prior to a 1 hr incubation at room temperature in 1 x TE/0.4mg per ml phenylmethylsulfonylfluoride (prepared as a 40mg/ml solution in propan-2-ol) to inactivate proteinase K and sarcosyl activity. Blocks were then either prepared for restriction endonuclease digestion or stored at 4°C in 0.5M EDTA
2.2.2(b). Restriction endonuclease digestion o f DNA in agarose blocks.
Prior to restriction enzyme digestion of DNA set in agarose, the blocks were washed for between 30 and 60 minutes in sterile distilled water followed by equilibration in the appropriate restriction enzyme buffer. Restriction enzyme buffers supplied by the manufacturers were supplemented with the addition of bovine serum albumin (to a final concentration of 4pg/ml) and spermidine (to a final concentration of40mM).
Half agarose blocks (50pl) were incubated in 15pl buffer, 80 to 85pl sterile distilled water and 20 units of enzyme (a final volume of 150pl), on ice for 30 minutes to allow complete enzyme diffusion into the agarose block. For complete digestion reaction mixtures were subsequently incubated for four hours at the appropriate temperature. When digesting with two enzymes, the agarose block was incubated with the first
enzyme for complete digestion, removed and washed for between 30 and 60 minutes with sterile distilled water followed by digestion with the second enzyme.
2.2.2(c). Pulsed field agarose gel electrophoresis.
Genomic DNA digested with rare cutting restriction enzymes was size fractionated by pulsed field gel electrophoresis using the hexagonal electrode array of the LKB- Pharmacia Pulsaphor apparatus. 150ml garose gels of between (of 0.8 to 1.2% w/y) were cast in 0.5 x TBE with 16 well combs. Blocks loaded into individual wells were sealed in with 1% LMP agarose to prevent them from floating out during electrophoresis. Gels were electrophoresed in 0.5 x TBE running buffer constantly circulating at 10°C using one of two sets of conditions described below, depending on the size of fragments to be resolved:
Pulse time (se c o n d s)
Time (hours) Voltage (V) Separation range (Kb) Program 1 Program 2 50 secs. 70 secs. 90 secs. 120 secs. 14 hours. 14 hours. 20 hours. 20 hours. 170V 170V 170 V 170V 50kb-900kb 200kb-1500kb
For size estimation commercially available yeast Saccharomyces Cerevisae
chromosomal DNA markers with a range between 200 and 2000kb, and phage Lambda concatomers with a range between 50 and SOOkb were electrophoresed along side digested genomic DNA.
2.2.2(d). Southern blotting of pulsed field agarose gels.
Following electrophoresis, pulsed field gels were stained in 0.5 x TBE containing Ipg/ml ethidium bromide for 15 minutes, before visualisation under ultra violet transillumination. Gels were then acid depurinated by submerging them in 0.25M HCl for 15 minutes and then treated with 0.4M NaOH for between 30 and 60 minutes. DNA was then transferred to Highbond N+ nylon membrane (Amersham) by capillary blotting through 3mm paper (Whatman) in 0.4M NaOH over a 48hour period.
2.2.2(e). H ybridisation and post-hybridisation washing.
Hybridisation of pulsed field membranes was performed at 65°C most usually in sealed plastic boxes and occasionally in bags as described previously (2.2.l.g.). Probes for hybridisation were radiolabelled and prepared prior to hybridisation also as previously described (2.2.l.f & g). The hybridisation technique is modified from that of Church and Gilbert (1984). Pre-hybridisation of membranes required incubation in 100ml of Hybridisation Buffer II. The larger hybridisation volume compared to bag hybridisation (2.2.l.g) required a threefold increase in the probe concentration added to the prehybridisation mix.
Post-hybridisation washing involved incubating in Church and Gilbert wash solution I (appendix 2A.4) at 65 °C for 2 x 15 minutes. If more stringent washes were required subsequent 15 minute incubations at 65°C in Church and Gilbert wash solution II (appendix 2A.4) were performed. Radioactive signal detection was as previously described (2.2. Ih.).
2.2.3. Yeast artificial chromosome (VAC) preparations.