DNA Storage and Quality
Reliable measurement of DNA concentration and purity is important for many applications in molecular biology, especially array comparative genomic hybridisation (aCGH) where accurate determination of DNA concentration is critical. Impurities in DNA can lead to inaccurate measurement of DNA concentration and could potentially inhibit subsequent labelling reactions. This article provides recommendations for measuring DNA quality for use in aCGH based on our experience of running thousands of samples per week in our high-throughput genomic services laboratory. In addition, detailed information is provided on the theory and practice behind measuring DNA purity and concentration.
The preservation and storage of DNA is of interest to scientists in a wide range of fields and disciplines. When considering what is meant by the terms ‘preservation’ and ‘storage’, it must be remembered that the perspectives of these scientists will vary considerably. For example, DNA required for testing a pharmaceutical product will need to be stable for a few years, whereas samples used in evolutionary biology have been in existence for millions of years1. The downstream applications in which these samples may be used are very different and therefore the minimum quality and quantity of DNA required will vary. Although nuclease contamination must always be carefully avoided when handling DNA, it is chemical degradation that represents the major threat to DNA preservation.
DNA storage strategies
In general, there are four broad strategies for long-term DNA preservation:
- Room temperature on a ‘dry’ solid matrix
- –196°C (storage in liquid nitrogen)
Two of these methods, dried and stored at room temperature and storage at –196°C, share a common mechanism where the DNA is maintained in a glassy (or vitreous) state. In the glassy state, molecules lose the ability to diffuse such that the movement of a proton is estimated to be approximately one atomic diameter in 200 years, thereby preventing chemical and nuclease degradation. If moisture is added to the ‘dry state’ or the temperature is raised above the glass transition temperature of water, movement and reactivity of protons is re-established and damage to the DNA can occur2.
The glassy state
Glass is a state of matter; glasses combine some properties of crystals with some properties of liquids. Glass formation, or vitrification, is the creation of a liquid solution with the viscosity of a solid. Glasses can be formed either by increasing the solution concentration or by lowering temperature. In frozen aqueous samples, glasses are formed by a combination of the two. Glasses are usually supersaturated and thus metastable, but the high viscosities and activation energies required for phase separation may prevent decomposition for long periods, the duration being dependent upon composition and temperature. The formation of glasses is normal for substances that remain liquid over a wide temperature range (the 'good glassformers') and can be induced for most liquids if cooling is fast enough to bypass crystallization. During reheating, but still below the melting point, good glassformers exhibit glass transitions as they abruptly transform into supercooled liquids, whereas other substances transform directly from the glassy to the crystalline state3.
Storage at –20°C to –80°C may well provide adequate conditions depending on the quality and quantity of DNA desired and the time frame in which the sample will be stored. However, neither of these conditions will maintain DNA quality equivalent to maintenance at liquid nitrogen temperatures over extended time periods (e.g. decades).
In contrast to storage of DNA in solution at very low temperatures, it is also possible to store DNA dried. This can be a practical alternative for long-term storage. In addition to reducing molecular mobility, dehydration also removes water that can participate in hydrolytic reactions. There are several methods of removing water from liquid preparations; these include spray drying, spray freeze drying, air drying or lyophilisation. Spraying DNA is perhaps the least popular option as it has been associated with damage introduced by shear stress.
Another option for storage of dried DNA is on FTA® Cards (Whatman). Cells are lysed upon application to the card and the nucleic acids are immobilized. Studies have shown that genomic DNA that has been stored on FTA Card at room temperature for over 17 years has been successfully amplified by PCR4. The cards are supplied with a reagent which enables high molecular weight DNA to be released from the matrix for use in many molecular biology techniques. Although DNA stored on FTA Cards may be suitable for microarray studies, this has not been tested by OGT.
In a laboratory setting, DNA is most commonly stored at 4°C, –20°C or –80°C. To avoid chemical and enzymatic degradation, DNA is often stored as a precipitate in ethanol at –80°C. Under these conditions, nucleic acids are stable for prolonged periods, but must be isolated from the ethanol, transferred to aqueous buffers, and typically quantified prior to use. These manipulations render ethanol precipitations undesirable for applications where the samples are needed on a regular basis. Aqueous solutions of DNA would be the most convenient, but nucleic acids are sensitive to depurination, depyrimidination, deamination and hydrolytic cleavage, which limit prolonged storage under these conditions. It is possible to inhibit these acid-catalysed degradation processes by storage in alkaline solutions. The ionic strength of the solution will also affect depurination rates, so storage in salt solutions as opposed to a low ionic strength buffer will help. Assuming the absence of nucleases when DNA is stored in a saline solution with pH8.5, the most common form of damage is via oxidation.
The rate of oxidation is enhanced by the presence of trace metals (e.g. Fe3+, Cu2+) due to the production of free radicals via Fenton-type reactions5.
The rate of oxidation is enhanced by the presence of trace metals (e.g. Fe³+, Cu²+) due to the production of free radicals via Fenton-type reactions4.
The Fenton reaction
The Fenton reaction was discovered in 1894 by H.J.H. Fenton. This reaction describes the breakdown of hydrogen peroxide by metal ions such as iron. The exact mechanism is still to be elucidated but, broadly speaking, the reaction can be described as:
Fe²+ + H2O2 → Fe³+ + .OH + OH-
Fe³+ + H2O2 → Fe²+ + .OOH + H+
The highly reactive free radicals that are produced during this reaction will go on to react with other compounds and can be a major cause of damage to DNA or other nucleic acids6.
Contamination by transition metals can increase oxidation levels; therefore, de-metalation of all components (DNA, buffers, and water) can significantly reduce degradation during storage. Unfortunately, is it impossible to measure very low levels of metal contamination or to completely remove trace amounts. Highly purified clinical grade DNA can contain iron levels of 30–40 ppb, which is well above the safe level of 5 ppb. A solution could be to incorporate chelators into DNA preparations to limit metal-catalysed reactions. But it should be remembered that chelating agents can inhibit nucleases which can have an adverse effect on downstream reactions. Also chelation of metals does not completely prevent Fenton-type chemistry and it may be advantageous to include antioxidants or scavengers in the storage medium.
A study by Qiagen7 showed that, following extraction using their QIAamp® DNA Blood Mini Kit, DNA was stable for at least 8 years; however, the quality of the DNA was dependent on the temperature and the buffering conditions used. DNA eluted and stored in Buffer AE (10 mM Tris·Cl; 0.5 mM EDTA, pH 9.0) was stable for at least 8 years at either –20°C or 2–8°C. DNA samples stored in water for 8 years remained intact when stored at –20°C, but were degraded to varying degrees when stored at 2–8°C. The degradation of the DNA stored in water at 2–8°C may have been due to acid hydrolysis (at pH 5–6), since water is unbuffered and can be slightly acidic. Alternatively, the DNA may have been eluted with water from a water source contaminated with minor traces of nucleases, or the DNA may have been contaminated with nucleases during the annual opening of the sample tubes. Buffer AE, supplied by Qiagen is intended to protect DNA during storage, with the Tris buffering against low pH and the EDTA inhibiting nucleases.
If extracting DNA from whole blood using the Gentra Puregene® Blood Kit, reports from Qiagen show that it is preferential to collect the sample in EDTA tubes to reduce DNase activity8. Heparin can be used but is not ideal as it is thought to bind to DNA during purification. Studies have shown that blood can be stored for up to 24 hours at room temperature prior to purification but, if possible, it should be placed on ice. For longer term storage, blood should be stored at 4°C or –80°C, studies have shown that good quality DNA can be obtained from blood stored at –80°C9. Storage at –20°C is not recommended as this can result in lower yields. The thawing temperature can also have an effect on DNA quality and Qiagen recommend that samples are thawed quickly at 37°C rather than slowly at room temperature10.
There is some controversy regarding DNA damage resulting from repeated cycles of freeze-thawing and it is common practice to store DNA in aliquots to minimise the number of times the DNA is thawed. Interestingly, a study by Bethesda Research Laboratories11 found no evidence that freeze-thawing cycles causes DNA damage. They made the observation that previous studies examining the effect of repeated freezing and thawing used radio-labelled DNA and the radioactive label itself was causing damage to the DNA rather than the number of freeze thaw cycles. However, as a cautionary note, this study utilised high quality DNA. Storing aliquots of DNA would still be beneficial in order to minimise degradation, which could occur at higher temperatures due to impurities, and to avoid contaminating the main DNA stock.
To ensure high quality microarray results, we recommend the following DNA storage strategies:
- Short-term storage (weeks) at 4°C in Tris-EDTA
- Medium-term storage (months) at –80°C in Tris-EDTA
- Long-term storage (years) at as –80°C as a precipitate under ethanol
- Long-terms storage (decades) at –164°C or dried
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