Extraction of DNA Using Monoclonal Anti-DNA and Magnetic Beads

There are many methods for extracting DNA before amplification by the polymerase chain reaction (PCR). Each has advantages and disadvantages, and all work reasonably well on relatively large quantities of fresh material. However, difficulties are encountered when the specimen is small or in poor condition (as is often the case with forensic and archaeological material) or contains inhibitors of PCR. To overcome these problems, an immunoaffinity method has been developed. It uses monoclonal anti-DNA bound to paramagnetic beads and is extremely efficient in extracting DNA at low concentrations. Also, the beads carrying the DNA/anti-DNA complexes can be added directly to a PCR mixture without elution, and the presence of any PCR inhibitors in specimens has no effect on amplification. Spleen cells were harvested from an (NZW × NZB) F1 mouse suffering from murine systemic lupus erythematosus (2), a condition characterized by the development of autoantibodies to DNA. The cells were hybridized with mouse myeloma cells to produce a monoclonal antibody of IgG2A class that demonstrated anti-double-stranded (ds)DNA specificity by the Crithidia luciliae dsDNA test (The Binding Site, Birmingham, England, UK). Using enzyme-linked immunosorbent assay (ELISA) techniques, the antibody was shown to react strongly with DNA from a variety of species (human, bovine and salmon) and weakly with plasmid DNA and oligonucleotides of approximately 20-mer in length. It also had weak but similar reactivity against 20-mer oligonucleotides of both GCGCGC... and ATATAT... composition. Initial studies showed that the antibody reacted well at 20°C, with optimal binding occurring in the presence of 1% Nonidet P-40 (Fisons Scientific Equipment, Loughborough, England, UK) and 145 mM NaCl; protein concentrations of greater than 5 mg/mL tended to inhibit the reaction. The antibody was bound to