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
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O'Brien, D. (2002). High-throughput DNA purification. Modern Drug Discovery. 5(3), 25-26.
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Kasper, Y. et Lenz, C. (2004). Stable 8-year storage of DNA purified with the QIAamp DNA Blood Mini Kit. QIAGEN News. 2004 e10.
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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.
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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.
Saliva is one of the most accessible of our body's bio-fluids making saliva sample collection easy and non-invasive. Saliva also harbours a wide spectrum of genetic data that can be used for genetic research and clinical diagnostic applications. It might surprise you to know that much confusion surrounds the source of genomic DNA in saliva. It certainly came as a surprise to me when I met with a number of customers on a recent trip across the continent.
In recent years, we've seen a marked increase in the desire to understand the genetic basis of disease as a means to improve patient diagnosis and treatment. While the range of research spans the spectrum of known diseases, all genetic research projects share one vital building block in that they require DNA as a starting point. Traditionally, DNA has been extracted from white blood cells extracted from whole blood. White blood cells are an excellent source of large amounts of high quality genomic DNA. However, because of the invasiveness and cost of obtaining, transporting and processing blood samples, researchers and clinicians have long searched for alternative methods. Over the past few years, saliva has become recognized as a very important and reliable alternative to blood samples for genetic research, clinical diagnostics, pharmacogenomics and more. What exactly is it that makes saliva such a good alternative to blood for genetic applications? It all comes down to the source of DNA in saliva.
Surprisingly, many people I spoke with assumed the source of DNA in saliva is strictly buccal epithelial cells, however, studies show that up to 74%(1) of the DNA in saliva comes from white blood cells. Yielding virtually the same amount of DNA per volume and the same DNA quality, saliva can be considered as good and as reliable a source of DNA for a wide variety of genetic applications. One thing to note however is that not all oral samples are equal.
Oral Sample Collection Methods
There are three methods for collecting oral DNA samples - dry, wet and non-invasive procedures.
Dry procedures require the donor to insert a cytobrush, buccal swabs or other collection device into the mouth where tissue is scraped from the gum and cheek surfaces. These methods collect primarily buccal cells that are lower quality and are potentially contaminated with bacteria from the teeth and other surfaces.
Wet procedures include swishing liquids in the mouth and spitting them into a collecting vessel. Mouthwash, which can contain a high percentage of alcohol content, is typically used for this procedure. The protocol, which can request the donor to swish for up to one minute, can burn and be uncomfortable for the donor. Mouthwash is also designed to remove bacteria from teeth and other mouth surfaces which results in a high amount of bacterial content being released into the sample.
Both the dry and wet methods described above do not prevent bacteria from growing in the sample and do not actively stabilize DNA. These methods also involve the insertion of an object or substance into the mouth. While it is arguably less invasive than venipuncture, it does not quite meet the definition of ‘non-invasive'.
Completely non-invasive collection using Oragene•DNA is a simple, painless procedure that requires the donor to spit into a collection device. After providing a sample and closing the device, a solution is released from the cap to mix with the saliva. This solution stabilizes the DNA for long-term storage at room temperature and prevents bacterial contamination. There is no requirement to insert an object or substance into the mouth - the user simply holds the tube and spits into it. In my opinion, that's the true definition of non-invasive DNA collection. As we like to say at DNA Genotek, "Just Spit, That's It!"
The high quality and high quantity of DNA collected with Oragene•DNA provides an excellent option for applications ranging from the largest molecular epidemiologic studies in remote locations, to bio-banking or HLA typing and even a single personalized medicine test.
Did anything in this article surprise you? Leave us a comment and let us know.
Interested in learning more about non-invasive DNA sample collection with Oragene•DNA? Register for our live webinar on April 21, 2010 at 11:00am ET.
References: (1) Thiede, C. et al. Buccal swabs but not mouthwash samples can be used to obtain pretransplant DNA fingerprints from recipients of allogeneic bone marrow transplant. (2000). Bone Marrow Transplantation. 25(5): 575-577.)