PCR amplification of specific DNA sequences from routinely fixed chromosomal spreads.

The growing field of application of the PCR prompted us to adapt this methodology as a routine screening for cytogenetic preparations. Cytogenetic laboratories usually store fixed cell pellets in fixative (methanol/glacial acetic acid: 3/1) a t 20°C. DNA from these fixed cell pellets can be extracted and analysed (1). However, for a large number of samples, fixed metaphase spreads on glass slides are only available. We report here that DNA isolated from fixed chromosomal spreads, stored at room temperature approximatively 1-5 years, is suitable for PCR analysis in spite of acidic depurination. Chromosome preparations were obtained as previously described (2). In some cases, fixed chromosomal spreads were G or R banded. In order to improve the DNA recovery, chromosome spreads were not scraped from the glass slides using a razor blade, but the slides were treated directly with proteinase K (0.5 mg/ml) in 50 yl extraction buffer (100 mM NaCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% SDS) under a sterile coverslip sealed with rubber cement at 55°C for 60-90 min. The coverslip was gently removed and the slide rinsed briefly with 100 /tl extraction buffer alone. After two extractions with phenol/chloroform and chloroform, the aqueous phase was then concentrated and purified from remaining chloroform, salts, and other small molecules by Centricon 30 microconcentrator (AMICON Inc., Danvers, MA). This last step improves the recovery of chromosomal DNA and allows the components of the extracts to remain in solution. Exons 7 8 of the P53 gene (3) were amplified from 10 /tl of the extract in 10 mM Tris pH 9/1.5 mM MgCl2/50 mM KCl/0.01% gelatin/ 0.2 mM dXTP/1 /*M of each primers (5'AGGTTGGCTCTGACTGTACC-3' and 5'-TTGTCCTGCTTGCTTACCTC-3') in 50 /il volume containing 2.5 units of Taq polymerase (Boehringer-Mannheim) for 30 cycles of 94°C denaturation (1 min), 58°C annealing (1 min) and 72°C extension (3 min) in an automated thermal cycler. After electrophoresis of 10 /d of the final PCR product on a 1.5% agarose gel, a specific 600 bp fragment of P53 gene was obtained as shown on the figure: (lane 1) unstained and unhanded chromosome spreads (lane 2) RHG-banded chromosomal spreads (lane 3) in order to monitor contamination, control extraction on glass slides without chromosomal spreads or cells (lane 4) 123 bp DNA ladder (BRL). We performed successfully the same study in fixed chromosome spreads, stored 1 5 years at room temperature. These observations indicate that DNA depurination (4) caused by acid fixation of metaphase chromosome does not prevent the PCR amplification of specific DNA sequences from relatively ancient cytogenetic preparations. Our procedure can be performed easily from routinely prepared and stored chromosome spread slides, and can be useful in retrospective studies when additional DNAs are not available for molecular analysis.