DNA Genotek, Ottawa, Ontario, Canada
Storage of specimens by refrigeration or freezing can add significant costs to a project. Oragene eliminates these costs by allowing saliva specimens to be stored for years at room temperature without DNA degradation.
Large population-based studies, involving hundreds to thousands of subjects, are frequently used to investigate the genetic determinants of complex diseases. Saliva is a convenient source of genomic DNA because it can be collected in a painless and non-invasive manner. For logistic reasons, samples often need to be stored prior to the extraction of DNA. Common storage methods, such as refrigeration and freezing, can add significant costs and inconvenience to a genetic study. In addition, these methods may be of limited effectiveness. For example, Ng et al. (2004) observed as much as a 30% decrease in PCR product yield from saliva samples that had been stored at -70C for 1 month.
An ideal protocol would allow saliva samples to be stored at room temperature for long periods of time with no DNA degradation. Oragene is a kit that collects and preserves DNA from saliva. This technical bulletin reports the ability of Oragene to stabilize and preserve DNA in saliva at temperatures up to 50C.
Materials and Methods
Oragene/saliva samples were collected and stored at room temperature (24C), 37C, and 50C for up to 187 days. At various time intervals, aliquots were removed and processed by the standard Oragene protocol. 200 ng of DNA from each sample was analyzed by agarose gel electrophoresis with ethidium bromide staining. The molecular weight of the extracted DNA was determined by comparison with a Lambda-Hind III digest ladder.
DNA yield was determined by the highly specific Fluorescence/DNase method (ref. 2, 3). The F/D method quantitates DNA using SYBR Green I dye (Molecular Probes, Inc.) and DNase treatment.
DNA from Oragene/saliva samples stored at 24C and 37C had a molecular weight > 23,000 bp and showed no evidence of degradation at various time points (Figure 1). Samples stored at 50C showed minimal degradation at 187 days. There was no change in the yield of DNA at 24C, 37C and 50C as determined by the F/D method.
Discussion and Conclusions
The rate of a typical chemical reaction decreases by half for every 10C decrease in temperature. Thus, it is expected that the degradation rate at 24C should be 5-fold less than the degradation rate at 50C. Since Oragene saliva samples remain stable for 187 days at 50C, they are expected to remain stable for 5 times as long at 24C (935 days or 30 months).
After 187 days at 50C, most of the DNA extracted from Oragene / saliva samples had a molecular weight > 23,000 bp. Since PCR is effective with DNA templates much smaller than 23,000 bp, it should take significantly longer than 30 months at room temperature for the DNA in Oragene samples to degrade to an unusable size. In summary, Oragene/ saliva samples are expected to be stable at room temperature for many years.
1. Ng, D., Koh, D., Choo, S., Ng, V., and Fu, Q. (2004) Effect of storage conditions on the extraction of PCR-quality genomic DNA from saliva. Clinica Chimica Acta. 343, 191-194.
2. Ballard, R., Anderson, M., Birkett, N., Gorn-Hundermann, I., Hansen, M., and Birnboim, H. (2004) Saliva as a source of large amounts of genomic DNA for population-based studies. Submitted for publication.
3. DNA Genotek Technical Bulletin PR003 Sept 30, DNA Quantifi cation Using the Fluorescence/DNase (F/D) Assay. 2004.
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