After size separation by electrophoresis in agarose gels, the DNA was transferred to nylon filters (Hybond N+, Amersham) by alkali transfer as previously described (Sambrook et a l, 1989). Gels of genomic DNA digests were initially nicked with 0.25M HCL for 10-15 minutes to improve transfer of large fragments, washed with distilled water and subsequently neutralised with 0.4M NaOH. Transfer was performed overnight and filters subsequently washed three times in 2x SSC, prior to storage at 4®C wrapped in Saran wrap.
Chapter two: Materials and methods 2.9.2 Radioactive labelling of DNA probes
DNA probes for hybridisation were labelled by one of two techniques. Generally, oligo- labelling was used; however for short oligonucleotide probes, end-labelling was the chosen technique.
Generation o f probes
DNA used as probes included fragments derived from restriction enzyme digestion of plasmid DNA. Digested DNA was electrophoresed through 0.8-1% low melting point (LMP) agarose; appropriate fragments were excised under UV visualisation, resuspended in an equal volume of TE and stored at 4°C. Single-band PCR products generated from genomic or cDNA were also used as probes. PCR products were diluted in sterile water (dependent upon band intensity) and 30pl purified by spinning down Microspin™ S-400 HR columns (Pharmacia Biotech) to remove excess primers and dNTPs in accordance with the manufacturer's instructions. Oligonucleotide probes were prepared as described in section 2.1.3.
Oligo-labelling of DNA probes
DNA probes generated to check the specificity of PML-RARa and RARa-PML PCR amplification products and for use in DNase 1 hypersensitivity studies of the CD2 gene were labelled with [a^^P]dCTP (Amersham International) by the random priming method (Feinberg & Vogelstein, 1984). 30-50ng of DNA was boiled for 5 minutes in the correct volume of distilled water for a total reaction volume of 50|il. When DNA fragments in LMP agarose were used, this step was preceded by heating of probe DNA at 65°C for 10 minutes to melt the agarose; the DNA volume did not exceed 25% of the total volume and if necessary a lOOpl reaction volume was selected. After brief spinning and placement on ice, for a 50pl reaction : lOpl 5x oligo-labelling buffer (OLE), l.Opl ESA (lOmg/ml) and 2.5pl [a-^^PJdCTP (25pCi) were added, followed by l.Opl DNA polymerase Klenow fragment (lU/pl) and mixed gently, prior to incubation for at least 2 hours at 37°C or overnight (15 hours) at room temperature. The reaction was stopped by addition of 50pl TE 8; unincorporated nucleotide was subsequently removed by spinning through a Sephadex 050 column. Labelling was considered satisfactory with at least 500 counts/second/pl. Probes lacking repetitive sequences were subsequently denatured with IM NaOH for 5 minutes at room temperature (volume added = 25% volume of probe), prior to addition to the hybridisation solution. For probes containing repetitive sequences, labelled DNA was boiled in the presence of 20pl (198pg) human placental
Chapter two: Materials and methods DNA (Sigma) for 5 minutes and then incubated at 65°C for 1-2 hours before adding to the hybridisation solution.
End-labelling
PCR products derived from cases of APL with PML/RARa rearrangements associated with disruption within the 3' region of PML were hybridised with an oligonucleotide probe spanning the RARa exon 3-PML exon 6 junction (5'-TCTCAATGGCTGCCTC-3') in order to distinguish bcr 1 and bcr 2 breakpoints, using a method based on that described by Gallagher et al. (1995). For a 50pl reaction, l|xg oligonucleotide was end- labelled with 30|iCi [y-32p] dATP (Amersham International) in the presence of 5pl lOx polynucleotide kinase (PNK) buffer (Promega) and 2pl T4 PNK (Promega). The reaction mixture was incubated at 37®C for 2 hours; unincorporated nucleotide was subsequently removed by spinning down a sephadex G25 column. Labelling was considered satisfactory with at least 500 counts/second/^il. No dénaturation step was required and probes were subsequently added directly to the hybridisation solution.
2.9.3 Prehybridisation and hybridisation of DNA probes to filters
Filters were pretreated with hybridisation solution in bottles (Hybaid, UK) for at least 2 hours at 65°C. After discarding the prehybridisation solution, filters were hybridised with labelled probe added to 20-25mls hybridisation solution overnight at 65°C. Filters were washed for 15 minute intervals at 65®C, the first wash comprised Ix SSC, 0.1% SDS; filters from DNase I hypersensitivity assays were subsequently washed to final stringency of 0.5x-0.2x SSC, 0.1% SDS according to the degree of radioactive signal, whilst filters derived from RT-PCR experiments were washed to a final stringency of O.lx SSC, 0.1% SDS. Filters from RT-PCR experiments were exposed to Kodak XAR or Fuji RX film for 1-12 hours, between intensifying screens at -70®C. For DNase I hypersensitivity experiments Kodak XAR film was used with exposure times of 3-14 days.
2.9.4 Prehybridisation and hybridisation of oligonucleotide probes to filters
Procedures were as described above (section 2.9.3), however prehybridisation and hybridisation were performed at 42®C. Filters were then washed in 6x SSC, 0.1% SDS for 30 minute intervals initially at 42®C, to a final temperature of 51®C prior to exposure to Kodak XAR film between intensifying screens for 1.5-3 hours at -70®C.
Chapter two: Materials and methods 2.10. Northern analysis
2.10.1 Electrophoretic size separation of RNA
Samples of total RNA (2pg) were diluted in lOpl RNA loading buffer, heated at 65°C for 5 minutes, then cooled on ice prior to addition of 4|il RNA loading dye. RNA was then separated electrophoretically through 1% agarose gels incorporating 1% formaldehyde in Ix RNA gel running buffer. RNA was visualised by staining the gel with ethidium bromide (0.5pg/ml). RNA was subsequently transferred to Hybond N (Amersham International) using 20x SSC; filters were then washed in 2x SSC and stored at 4°C in Saran wrap.
2.10.2 Prehybridisation and hybridisation of Northern filters
Filters were prehybridised using 20mls prehybridisation solution (for Northern analysis, see section 2.17) for 24 hours at 42°C, this was then replaced with fresh hybridisation solution containing the radiolabelled DNA probe. These were labelled as described in section 2.9.2; probes were denatured by boiling for 5 minutes prior to addition to the hybridisation solution which had been preheated to 42°C. Filters were then hybridised at 42°C for 18-19 hours.
2.10.3 Washing of Northern filters
Filters were washed twice (10 minutes each wash) in 2x SSC, 0.1% SDS at room temperature, followed by a final 20 minute wash in O.lx SSC, 0.1% SDS at 50°C. Filters were then shaken to remove excess wash solution, wrapped in Saran wrap and exposed to Kodak XAR film between intensifying screens at -70°C for at least 1 week.