Extensive guidelines for preserving specimen or tissue for later DNA ...

Extensive guidelines for preserving specimen or tissuefor later DNA workv1.0 by Camiel Doorenweerd & Kevin Beentjes Jan 2012.Degradation processes set in the moment an organism dies. Cell membranes begin to break down and allsorts of enzymes and chemicals previously safely stored inside the cells are free and start to break downorganic compounds, including DNA. Outside threats, such as bacteria and moulds, now also have free entryto all of the cell’s precious content. DNA molecules are relatively strong, but their length makes them weakand they are degraded by cutting them into smaller sections. This continues until the sections become sosmall they can longer be called DNA. The speed of the process depends on many factors, in general when itcomes to preserving tissue for later DNA work:Cold and dry is good, warm and wet is bad.The complexity with preserving specimen and tissue for DNA work comes from the fact that there are noabsolute consequences of the methods (with some exceptions mentioned below), but there will be a gradualeffect in time on the success rate with later DNA work. Preserving DNA is thus a matter of ruling outdamaging factors as much as possible. If it is not possible to keep your specimens dry, at least try to keepthem cold, and vice versa.PreservationCold versus warmMost enzymes have an optimum working temperature around 37-40°C, and chemical reactions in generalwork faster with high temperatures. Above 65°C the double stranded DNA helix loses its bond and becomessingle stranded (called denaturation of the DNA), making it more vulnerable. For preserving DNA it really is:the colder the better. The lower the temperature the more the chemical reactions will be slowed down andDNA will be preserved (also explaining why DNA can be recovered from Neanderthals trapped inpermafrost). However, care should be taken with freezing and thawing cycles. During thawing, minute icecrystals can emerge and create damage as well. Thus, only if specimen are to be stored for a long time it isrecommended to keep them frozen (-20°C in a normal freezer, -80°C in an ultra freezer or -180°C at liquidnitrogen), if specimens still need to be examined or if the box they are stored in needs to be used frequently;keep them in a fridge at around +4°C.Dry vs wetWater mediates transport of enzymes and chemicals, and is an excellent medium for many bacteria andmoulds that will degrade DNA. Dehydrating, removing water, will thus preserve DNA. Highly humidconditions, such as rainforests, are often problematic for preserving DNA. In these situations specialmeasures need to be taken.Liquid NitrogenIdeally, one would go out in the field with a can of liquid nitrogen (-180C) and kill and preserve specimens inthat can. This will often be impractical for many reasons, dangerous, and not to mention expensive withliquid nitrogen costing around €20 per litre. It is therefore not often used, but theoretically ideal for DNA.EthanolEthanol (EtOH) can be a killing agent as well as a preservative, but the percentage is critical. Ethanol drivesout water from tissue and cells, thus dehydrates the tissue and so preserves DNA. Ethanol 96% (indicatedhere as 96, but can be 94-100%) is an excellent killing agent and preservative, and is ideally combined withcold for optimum preserving conditions for keeping DNA preserved for over a hundred years.70% ethanol is often used instead of 96% because it is cheaper and keeps specimens somewhat flexible,allowing for later morphological study. 70% ethanol however also means that there is 30% of other things inthis liquid (mostly water), and this causes degradation of DNA. DNA is actually better preserved with dry(pinned) specimens, than with material stored at 70% ethanol. In general tissue will remain ok for DNA workwith 1-2 years stored in 70% ethanol, whereas dry specimen can be used up to 10 years old with a similarsuccess rate.Care must be taken not to use methylated ethanol. Ethanol is quite popular for human consumption andgovernments put heavy taxes on it. 70% or 96% ethanol that is not meant for consumption is often madeunsuitable for consumption by methylating it (adding methyl groups). It then becomes poisonous for humans,but also harmful for DNA. In developing countries it will often not be indicated on the label! Methylated

ethanol can quite easily be recognized by its smell. Always use laboratory quality non-methylated 96%ethanol.The volume ratio of specimen and ethanol is also important. Organisms consist of up to 70% of water. If forexample a bumblebee is stuffed into a 1.5ml container with 1ml 96% ethanol, the end concentration of theethanol will be much lower. In such cases a larger container must be used, or the ethanol needs to berefreshed several times before long term storage. The same goes for organisms that live in water (salt orsweet), as they are especially wet.SilicagelSilicagel is mostly used by botanists to dry and preserve plant material. It is a salt that will be supplied insmall balls or grains that absorb water from the air and contain it. There are many different typescommercially available, with different grain sizes (small grains have a larger surface area thus absorb faster)and optionally with a colour that will change once the salt is saturated. This then indicates that more silicagelneeds to be added or the whole set needs to be changed. The blue-purple indicator silicagel is still popular,but the cobalt that is used in these is toxic, and it is now more and more being replaced with a non-toxicorange-transparent indicator. A combination of small grain non indicator silicagel with some indicatorsilicagel (generally larger grains) is ideal. Be sure to use enough and keep an eye on the indicator. To becompletely sure of good preservation of DNA the samples can be frozen with the silicagel.Saturated silicagel can be re-used by drying it in a pan or oven at around 100-140°C. Microwave can beused, but if the silicagel gets too hot the particles will melt together, making it quite useless.DMSODMSO, or more accurately DMSO salt solution, is a solution with saturated salt levels. Specimen can bepreserved with this when for example ethanol is problematic, and has mainly been used with marine animals.Even though this medium will preserve the DNA well, problems will arise when using ethanol based DNAextraction methods. The DMSO salt will react with the ethanol creating a foam and irreversible damage tothe DNA. As most DNA extraction methods use at least one ethanol step, it is better to avoid thispreservative. It should also be noted that when the specimen is taken out of the solution it will have a layer ofsalt on it that takes many rinsing steps to remove.FTAFTA is a membrane developed by the company Whatmann in the 1980’s. It is supplied in the form of a smallcard, with one or up to 96 circular membranes where (soft) tissue can be placed to preserve its DNA. Themembrane will secure the DNA and when it is stored in dry conditions will be stable for at least 30 years(they haven’t been able to test it longer). The cards can be purchased with a colour indicator, which will turnfrom pink to white when matter has been applied successfully. The cards are quite expensive and there isonly room for a small amount of tissue on the cards, plus it is not easy to process many samples with DNAwork later. The cards are mainly useful in situations where Ethanol cannot be used, or where samples needto be shipped by post.Absolute Don’tsSpirits - Contains highly methylated ethanol and other harmful chemicals.Formalin - Affects DNA in such a way that it becomes impossible to work with.Killing agentsEthyl-AcetateCurrently used in nail-polish remover and as such available in any supermarket. It is a killing agent forarthropods used by applying a few drops to some cotton and placing it with the arthropod in a closedcontainer (often referred to as a killing-jar). Although the smell takes some getting used to, it is harmless tohumans. It has in the past been accused of causing damage to DNA. This has never been shown to be true,and high success rates are possible with specimen killed with ethyl-acetate. To be on the safe side however,specimen should not be left in the presence of ethyl-acetate (i.e. in a killing-jar) longer than necessary.CyanideA good alternative killing agent to ethyl-acetate that is used in a similar manner, but extreme care should betaken when handling this highly toxic (deadly to humans!) chemical.AmmoniaAmmonia is sometimes used for killing arthropods, mostly Lepidoptera, and used in a similar manner asethyl-acetate. There are no harmful effects on DNA known, but as with ethyl-acetate do not leave thespecimen longer in contact with the ammonia than necessary.2

AcetonePreviously used in nail-polish remover, but now only available in hardware stores as it is a quite aggressivechemical. It is often used by Odonatologists, as killing agent but also as preservative. As a killing agent thenegative effect on DNA seems to be negligible, but as a preservative there appears to be a large loss ofsuccess.Other issuesContaminationContamination occurs when the DNA of one sample ends up with another sample, causing the wrong DNAto be analyzed with the associated data. The risk of contamination needs to be estimated before starting andappropriate measures need to be taken.The risk of contamination is the largest when working with a combination of DNA-rich and DNA-poor tissueor specimen. Fresh material will contain much more DNA than old (i.e. museum) material, the small amountof DNA is then easily contaminated with the larger amount. When dealing with soft tissues, large amounts oftissue will similarly contain more DNA, and easily become a source of contamination. When working withchitinous insect parts, the risk of contamination is quite small if these parts remain intact.In general all sampling tools and the sampling location should be cleaned before starting and cleaned againin between sampling steps. If possible, clean everything with a 5-10% bleach solution (or a commerciallyDNA removal liquid), but never forget to rinse afterwards with (tap)water! Make sure all traces of bleach arerinsed off the tools, because these will highly effectively damage DNA if they remain present. Alternatively aUV-light can be used to sterilize the working space and equipment, but this will usually only be available in alaboratory environment. Heating tools to a high temperature, i.e. by using a Bunsen burner, will sterilize themas well but also cause coaling and damage to the tools.Working with gloves is good, but not absolutely necessary. Dead skin cells contain few DNA and it would besurprising to get contamination this way. Note that when working with RNA the story is very different,because skin is often covered with RNAse enzyme, which breaks down RNA. According to my molecularbiologist teacher in University this is especially so with people that drink milk.UV-light and Nuclear radiationUV-light is neither a killing agent or preservative, but should be kept in mind when preserving tissue for DNAwork. Direct exposure to UV-light is extremely harmful for DNA. Even though tissue will mostly be placed incontainers and liquids, it should never be left it out in the direct sun. All types of nuclear radiation are harmfulto DNA by causing mutations and other types of damage, but this will most hopefully not be a factor that youwill have to worry about during your collecting or processing of samples.Non-destructive DNA extractionPerhaps only remotely relevant for these guidelines, but it is good to be aware of its existence. With socallednon-destructive DNA extraction a piece of tissue is used that contains chitinous parts that can berescued and mounted on a slide (such as an abdomen containing genitalia). During the DNA extraction anenzyme is used that is called proteinase K. This enzyme breaks down cells to release DNA into solution, butwill only affect soft tissue. Chitinous parts that are often used for morphological study become more visibleafter the proteinase K step and can be mounted on a slide for further study. In this way the DNA is extracted,but the sample remains available for morphological study as well. This is a highly laborious process, but itcan be worth it in some cases. It is always easier to preserve DNA as a pure DNA extract instead of tissue orspecimen that contain DNA. If you can choose between creating a DNA extract now rather than later, alwaysdo it now.