The cross-contamination of DNA templates during autoclaving and leakage of the contaminant from the autoclave have been suspected but not verified by reliable evidence (9). We used model waste composed of a piece of wipe-paper, PCR tubes, pipette tips, and 400 µL PCR product containing (7.15 ± 0.01) × 1014 copies of the template (mimicking typical laboratory waste contaminated with PCR products) for assessing DNA contamination from the autoclave. The model waste was carefully placed in a 500 mL Pyrex beaker and then located at the center of the stainless steel basket in the autoclave chamber so that the waste was not in contact with the surroundings but was in contact with the atmospheric phase over the wall of the beaker with a height of 4 cm.
After processing at 121°C for 20 min, (2.12 ± 0.42) × 106 copies amplifiable template (mean ± SD for 4 separate autoclavings) were found in the bottom water. More than 1/108 of the starting amount of model DNA molecules were considered to be discharged from the model waste and collected into the bottom water during autoclaving. Meanwhile, amplifiable template was not detected in the steam fractions after autoclaving the model waste. Since the lower limit of calibration for the real-time PCR assay was 8.10 copies/1 µL sample, the absence of amplification implies very little or no template in the steam (less than 810 copies in total), but it is obvious that the migration of template DNA from the model waste to the bottom water was mediated by the steam. Furthermore, steam fractions taken before cleaning the chamber were not free of pUC19 template, either. Therefore, steam may contain a trace amount of template DNA, contaminating the exhaust system. We conclude that autoclaving is a source of contamination that could result in false-positive PCR reactions. Thus, it would be better to dispose of DNA waste without autoclaving. However, as demonstrated by Gefrides, et al. (7), DNA is susceptible to extended processing by autoclaving. If the autoclave is utilized for decomposing DNA waste, contamination via exhaust steam has to be carefully avoided. For this reason, we endeavored to find an effective protocol for autoclaving DNA.Decomposition by encapsulation
We examined whether contamination by leakage could be prevented when autoclaving encapsulated DNA samples. Ten microliters of a PCR product containing (8.71 ± 1.15) × 1011 copies of the template DNA were encapsulated in a 2 mL polypropylene micro-tube with a screw-cap and an ethylene-propylene rubber O-r ing (ASSIST Tube 72.694.100S, SARSTEDT, Tokyo, Japan). The initial amounts of the samples and evaporation during autoclaving were assessed gravimetrically at a precision of 0.01 mg. The micro-tube was supported by a cubic tube rack with a height of 9 cm (USA Scientific, Inc., Ocala, FL) and placed at the center of the stainless steel basket. Sample leakage was not observed during the autoclaving. Figure 1 shows an electropherogram for the autoclaved samples. The band at 2682 bp disappeared, and a smeared band at around 100 bp was observed after 20 min and 40 min processing. The smeared band disappeared after 120 min of processing. PCR template activity was not observed in the 120 min processed sample, while it remained in the 20 min and 40 min samples. It has been proposed that the air must be removed from the autoclave chamber as sterilization capability has been proven only with saturated steam (1, 2). However, as shown here, the structure of DNA and amplifiable template could be eliminated by autoclaving even under encapsulation with air (i.e., without replacing the air with saturated steam).
Effect of unsaturated steam
Removal of template activity by autoclaving with encapsulation was further investigated using triplicate samples. A 10 µL aliquot of the sample was autoclaved, followed by 10-fold dilution with TE, and the 84 bp fragment was then quantified. In two of three tubes containing encapsulated PCR product [(8.27 ± 0.34) × 1011 copies/ tube; determined by triplicate panels of the digital PCR assays] no amplification could be detected following autoclaving at 121°Cfor 40 min, while one had (1.74 ± 0.13) × 104 copies/tube. More than 99.9999% of the template was considered to be removed by this treatment. Whereas amplifiable template appeared to be eliminated in the two samples, it is possible that the template DNA was present at quantities lower than the detection limit (8.10 copies/1 µL sample). Longer autoclaving times or dilution of DNA solutions prior to treatment (less than 108 copies/µL or 1 ng DNA/µL) will be desirable for complete decontamination or management of the environment for sensitive applications.