The Use of  -DNA as an Internal Standard in the Detection and Quantitation of DNA Damage in Specific Genes Using Southern Blotting (1996)

Modified Southern blotting assays are powerful techniques for analysing DNA damage and repair in specific genes caused by, for example, ultra-violet (UV) light (1), or the anti-cancer agents mechlorethamine (HN2) (2) and cisplatin (3). The assay relies upon the availability of enzymatic or chemical reactions which can convert damaged bases to single strand breaks. In the case of UV-induced pyrimidine dimers or cisplatin lesions, strand break formation is catalysed by the enzymes T4 endonuclease V and ABC excinuclease respectively (3,4). N-alkylpurines formed by HN2 are converted to strand breaks by neutral depurination followed by alkaline hydrolysis (2). In all these methodologies, DNA from treated cells is digested with a restriction enzyme, cleaved at damage sites, denatured, electrophoresed, Southern blotted and hybridised to a specific probe. DNA damage is determined as a function of the signal from treated DNA relative to an untreated control. When studying damage and repair it is essential that equivalent amounts of DNA are loaded into each well, because errors in DNA quantitation or gel loading will result in inaccurate measurements of DNA damage. To overcome this problem we have included the use of human α DNA as an internal control of both DNA extraction and subsequent electrophoresis and Southern transfer. As an example the method is described to detect HN2 damage in the N-ras gene of human tumour cells. Exponentially growing Colo320HSR cells were treated with HN2 in RPMI at 37 C for 30 min, washed in ice-cold PBS, and the DNA purified. DNA was extracted by a modification of Marmurs method (5), adapted for use with mammalian cells (6). Cells were lysed with 9 ml lysis buffer (400 mM Tris–HCl, pH 8.0, 60 mM EDTA, 150 mM NaCl, 1% w/v SDS) containing RNase (10 μg/ml) and incubated at 37 C for 30 min. Three millilitres of 5 M sodium perchlorate was added and the solution mixed by inversion for 20 min and incubated at 65 C for 20 min. The samples were cooled to room temperature and extracted with 15 ml chloroform. The DNA was precipitated with 2 vol ethanol at –20 C, washed twice in 70% ethanol, dried, and dissolved in water. DNA was digested with 1 U/μg EcoRI (50 U/μl; Life Technologies) for 1.5 h at 37 C. The DNA was extracted with 1 vol chloroform, ethanol precipitated, washed twice in 70% ethanol, dried, and dissolved in water. DNA concentration was determined by absorbance at 260 nm, and 30 μg samples of DNA in 30 μl of water were heated at 70 C for 30 min to depurinate alkylated bases. Apurinic sites were converted to strand breaks by addition of 3 μl freshly prepared 1.1 M NaOH for 30 min at 37 C (2). Four microlitres of 10× loading buffer (50% v/v glycerol, 0.04% bromophenol blue) were added, and the samples were size fractionated using a 0.5% agarose gel containing 0.05 μg/ml ethidium bromide in TAE (40 mM Tris, 2 mM EDTA, 20 mM sodium acetate, pH 8.1) at 2 V/cm for 16 h. The DNA was visualised under UV, and the gel was blotted onto Hybond N+ (Amersham) for 16 h in fresh 0.4 M NaOH using descending transfer (7). Filters were first hybridised with the insert of pAT8.8.7 containing the 5′ PvuII fragment of human N-ras labelled with [α-32P]ATP to detect the 8.8 kb EcoRI fragment of the human N-ras gene (8). Filters were washed twice with 2× SSPE, 0.1% SDS at 65 C for 15 min, then twice with 0.1× SSPE, 0.1% SDS at 65 C for 15 min and autoradiographed. Filters were then stripped according to manufacturer’s instructions, and the human α DNA was detected by rehybridisation of the stripped filter at 42 C with 32P-end-labelled oligonucleotide 5′-TCATAGAGCAGTTAGGA-3′ (Pharmacia). Filters were washed twice for 15 min at in 1× SSPE, 0.1% SDS at 42 C. After autoradiography, band intensities were determined using a BioRad scanning densitometer. When human genomic DNA is digested with EcoRI, α repeats which account for 1% of the human genome yield small 340 bp units (8). In Figure 1a it can be seen that when genomic DNA was treated in vitro with HN2, there is no appreciable loss of signal from the α band at 50 μM even though the signal has totally disappeared from the N-ras band. This is because α is 30 times smaller than N-ras, and so will receive on average 30-fold less damage. The same effect was observed when genomic DNA from Colo320 HSR cells treated with HN2 was used (Fig. 1b), although higher doses of drug are required to produce the same level of damage in cells compared with naked genomic DNA (9). The band intensities of duplicate samples of N-ras showed variation, particularly at low concentrations of HN2. The signal from track 5 showed a high signal on hybridisation with N-ras which is mirrored by a similar rise in α signal, presumably due to errors in DNA quantitation or gel loading. The levels of α from treated cells do not fall significantly below those of the untreated control at the concentrations of drug used which produce less than one lesion per N-ras fragment. The α signal was therefore used as the