× Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø

The practice of autoclaving reagents and supplies used in aseptic protocols has been widely accepted within the scientific community. There has been a question however, as to whether steam sterilization destroys all possible contaminating DNA-especially sequences synthesized by the Polymerase Chain Reaction (PCR). It has been proposed that the high pressures involved in autoclaving simply scatters the DNA molecules randomly about the vessel they are contained within rather than breaking them down. To address this question, an experiment was performed to determine if DNA subjected to steam sterilization would be broken apart to the point where it was unsuitable for amplification of the DQA1 and PM systems.

Three samples of approximately 40-50 ng of double stranded DNA as well as three samples of PCR product DNA were placed in GeneAmpÔ tubes. These were enclosed in a plastic box and autoclaved for three minutes at 273°C. Following autoclave treatment, 200ul of TE was placed in the sample tubes, vortexed and left overnight at room temperature. Samples were concentrated using Centricon 100 devices and 20 ul of each sample was used to amplify DQA1 and PM systems. Amplified samples were applied to 4% agarose gels made with 0.5X TBE buffer and 0.05 M EtBr to detect amplified product bands. The samples were then typed using a DQA1 + PM Amplitype provided by Perkin Elmer.

Two of the three double stranded DNA samples did not have detectable product bands when run on the gel. No interpretable typing results were obtained from these two samples. One of the previously unamplified samples did have product bands for four of the PM markers. The bands present in this sample corresponded to the GC, D7S8, HGBB and GYPA loci. Very faint allele dots were obtained for each of those loci on the typing strip, however there was no "S" control dot. Larger loci, LDLR and DQA1, were not amplified. In contrast, all three of the previously amplified samples had product bands corresponding to the six DQA1 and PM loci. Typing results for all of these samples matched the DQA1 and PM profiles which had been obtained from them in previous testing. These results prove amplified DNA products are more substantial than can be disposed of by autoclave sterlization.