A few weeks ago, I posted an article about best practices for long term storage of unpurified Oragene•DNA samples on The Genetic Link. The response to the article was positive so I'm following up with today's story about long term storage of DNA purified from Oragene•DNA samples.
The preservation and storage of DNA is an important consideration for molecular epidemiology and population studies. As the makers of the Oragene•DNA family of products, we have prepared these recommendations for the long-term storage of DNA purified from saliva samples collected with Oragene•DNA to help our customers achieve optimal results.
Preventing DNA Degradation
There are three major causes of DNA degradation in a purified sample (ref. 1). Samples may be accidentally contaminated by bacteria, but storage at 4°C or lower will minimize bacterial metabolism and the release of nucleases. DNases may be inadvertently introduced from the skin, but this can be minimized by wearing gloves when handling samples. Repeated cycles of freezing and thawing may also contribute to DNA degradation. This may be minimized by splitting the purified DNA into multiple aliquots and thawing one at a time.
There are two major causes of degradation of purified DNA:
- Acid hydrolysis - due to storage in a solution with no buffering capacity, explained in "Comparison of TE and water" section
- Nuclease activity - DNases may come from the skin of the person handling the sample, contaminated labware or bacterial contamination of the purified sample. Use of gloves can minimize contamination from the person and use of nuclease-free labware and working in a clean environment can prevent both nuclease and bacterial contamination. Additionally, inclusion of a metal ion-chelating agent, such as EDTA, in the storage buffer can effectively inhibit the activity of any DNases present in the sample.
Comparison of TE and water
Kasper and Lenz (ref. 2) performed an 8-year study of DNA stored in water or Buffer AE (10 mM TrisHCl; 0.5 mM EDTA, pH 9.0). DNA stored in Buffer AE at -20°C or 2-8°C showed no degradation by gel electrophoresis and amplified well in a PCR assay. DNA in water remained intact when stored at -20°C but samples were degraded when stored at 2-8°C and performed poorly in a PCR assay. Pure water lacks buffering capacity and an acidic pH may lead to DNA hydrolysis.
Biobank recommendations
An EU workshop on Biobanks (ref. 3) recommends freezing DNA samples to prevent bacterial contamination and to minimize evaporation of the sample. Tris-EDTA (TE) buffer contains sufficient buffering capacity to prevent acid hydrolysis of DNA. Similarly, the UK Biobank (ref. 4) recommends the storage of DNA in a nuclease-inhibiting environment at a temperature of -20°C, since no significant increase in stability is observed at temperatures below -20°C.
References
-
O'Brien, D. (2002). High-throughput DNA purification. Modern Drug Discovery. 5(3), 25-26.
-
Kasper, Y. et Lenz, C. (2004). Stable 8-year storage of DNA purified with the QIAamp DNA Blood Mini Kit. QIAGEN News. 2004 e10.
-
Biobanks for health: Optimising the use of European biobanks and health registries for research relevant to public health and combating disease. Report and recommendations from an EU workshop held at Voksenåsen Hotel, Oslo. January 28-31, 2003.
-
Sample handling and storage: Subgroup protocol and recommendations. Version 1.0. UK Biobank. July 7, 2004
We hope these best practices will help you optimize the long term storage of all your purified Oragene•DNA samples. If you like this article, be sure to let us know by leaving a comment.
The number of clinical trial and epidemiological studies collecting genomic DNA from a large number of individuals is increasing rapidly. There are many options for obtaining these biospecimens including blood collection, saliva collection, tissue and more. Yet, recruitment is perhaps the most challenging part of any scientific research study. Potential study participants are often reluctant to participate because they are needle phobic, do not want to travel to a specific location to participate in the collection process or are otherwise inconvenienced by the study criteria. Problems with recruitment can disrupt the timetable for a research project, preoccupy staff and, ultimately, result in a trial being abandoned (Ashery & McAuliffe, 1992).
At DNA Genotek, we set out to discover how several successful studies have been able to meet their recruitment and compliance goals in a timely way and to summarize their success criteria in a report. We accomplished this through telephone interviews and by reviewing published research. Many of your peers provided the kind of insight that previously had not existed. The result of this work is the research report titled: Overcoming challenges in DNA sample collection - How epidemiological researchers are maximizing compliance rates with non-invasive DNA self-collection.
This report will help you discover:
-
How successful researchers are achieving dramatically higher compliance rates (70.52% to 95% in the first phase of collection)
-
What processes (from qualifying participants to analyzing results) most contribute to higher compliance rates
-
Whether centralized or mail-based collection impacts compliance rates
-
How to overcome challenges to maximize compliance
-
How to take a good compliance rate and make it an outstanding compliance rate
- What issues most affect compliance rates
If you're considering starting a clinical trial or epidemiological study, it's our hope that this report will help you build the criteria for successful DNA collection and for maximizing your compliance rate. If you're already working on a study, feel free to examine what these experienced researchers are doing (and use this study to help others).
Want to learn more about how to meet your recruitment and compliance goals?
Download this free report to learn best practices for recruitment and compliance from top research organizations.
Would you like to see us publish more reports like this? Leave a comment and let us know.
This month, PLoS ONE published a new research study that I think will interest readers of The Genetic Link. The study, titled "Array-Based Whole-Genome Survey of Dog Saliva DNA Yields High Quality SNP Data", was written by Jennifer S. Yokoyama, Carolyn A. Erdman and Steven P. Hamilton of the Department of Psychiatry and Institute for Human Genetics, at the University of California, San Francisco. The study is interesting for several reasons.
-
The study highlights the availability of array-based genotyping platforms for canines but notes that they require large quantities of high quality genomic DNA.
- The authors state that there is an increasing demand in the research and clinical communities for larger sample studies to ensure statistical significance.
- The study notes that DNA sampling can become the limiting factor when studying canine behavioural traits.
The authors evaluate the performance of whole blood and Oragene•ANIMAL DNA collection kits for GWAS studies. DNA from all samples was analyzed using Illumina's Infinium Canine SNP20 genotyping array. The researchers' experience suggested that owners were more conducive to returning Oragene•ANIMAL kits than to agree to a blood sample. Surveys sent out with the kits also indicated that the owners found the collection to be very easy. The sample collection was successful for all participants and the process took less than 10 minutes.
High-throughput whole genome array genotyping was performed with outstanding results. The samples achieved very high overall genotype call rates (>99.5%) with very good concordance. They found that on average, genotypes from DNA derived from blood and saliva from a single dog matched >99.9% of the time*.
The researchers found the Oragene•ANIMAL DNA collection kits very favourable due to their ease-of-use and non-invasive approach, making them a preferred method of sample collection for future studies. According to the researchers: "Our results demonstrate that saliva collection from dogs is facile, convenient, and yields large amounts of high quality DNA that provide excellent performance on high-throughput whole genome arrays."
With Oragene•ANIMAL, researchers will be able to collect the number of samples required for GWAS studies and they can be confident of the quality and quantity of the DNA and its performance for array-based studies.
Want to read the full details on how Oragene•ANIMAL performed compared to whole blood in this study? Click here to link to the full report.
*Source: Yokoyama JS, Erdman CA, Hamilton SP (2010) Array-Based Whole-Genome Survey of Dog Saliva DNA Yields High Quality SNP Data. PLoS ONE 5(5): e10809. doi:10.1371/journal.pone.0010809