Ancient DNA: Methods and Protocols (4 page)

3.2. Reagents

One should assume that all reagents and tools are contaminated
and Materials

with human DNA, even those labeled as sterile. Although few products are certifi ed to be DNA-free, many are certifi ed as nuclease-free; using these is key so that what little aDNA remains in an extract is not degraded upon contact with nuclease-contaminated materials. For the greatest assurance that potential contaminants 8

T.L. Fulton

are removed, especially for human studies, all solutions should be fi lter sterilized.

All equipment must be decontaminated before use, for example by UV irradiation (45 W, 72 h), baking at 180°C for 12 h, acid-treatment with 2.5 M HCl for 48 h, or 3–5% bleach (up to 50%

dilution of store bleach; sodium hypochlorite) for 48 h
( 26, 35
) . It has been recommended that autoclaving is not used for steriliza-tion, as it breaks DNA into short (~100 bp) fragments and may increase the potential for bacterial contamination
( 35
) . However, many of the methods listed above may result in problems if they are not performed effectively. As all function to break down DNA, if the reaction is incomplete (i.e., not high enough chemical concentration; insuffi cient time of exposure to UV), the resulting fragmented, damaged, modern DNA appears and behaves as

aDNA. Though widely used, UV destruction of DNA mimics that of natural environmental processes, causing photoproducts in
DNA that lead primarily to C–T transitions ( 36
) . Bleach causes oxidative damage to DNA, producing chlorinated base products and cleaving DNA into progressively smaller fragments
( 37 )
. If this process is incomplete, modern contaminant DNA will be fragmented and appear ancient. Thus, it is important to adhere fully to decontamination procedures, so as not to disguise modern contamination as the targeted aDNA.

Studies of domestic animals should also consider that PCR

reagents are potentially contaminated not only by human DNA, but also by animal DNA
( 27 )
. Thus, negative controls including carrier DNA are important in both human and animal studies, as is considering the source of the reagents. For example, if ancient cattle or bison DNA is the target, rabbit serum albumin (RSA) may be a better choice than bovine serum albumin (BSA) in PCR.

3.3. Maintaining

To maintain a sterile working environment, all personnel should
a Sterile Space

wear a full body suit including a hood, mask, shoe covers, and gloves at all times. Personnel should not enter the lab unless they have showered and changed into clothing that has not been in the PCR lab (see Note 1). Every surface should be cleaned before and after any work is performed; an additional thorough weekly cleaning is also important to maintain a sterile work environment.

Surfaces can be cleaned in a number of ways to destroy any contaminant DNA present: 3–10% bleach followed by 70% ethanol (to clean away bleach and avoid corrosion), acid, and/or nightly UV

irradiation. Keep in mind that the same problems of insuffi cient decontamination apply as discussed in the previous section.

3.4. Sample

The most effective way to avoid contamination of samples is to
Preparation

adhere to stringent aDNA sterility protocols from the moment
and Storage

that the sample is excavated onward. Contamination at the point of collection is a serious problem, particularly for bones and teeth
( 38, 39 )
. However, it is not always possible to be certain of the 1 Setting Up an Ancient DNA Laboratory

9

collection and storage history of every specimen. Even those samples collected in the most sterile manner may still have surface contaminants from the depositional environment. Many protocols have been proposed that attempt to remove surface contaminants from ancient bones and teeth. These include the physical removal of the outer surface, washing or prolonged exposure of the outer surface to chemicals including water, EDTA, bleach, ethanol, acid, or hydrogen peroxide, UV irradiation of the sample, and/or extrac-

tion of the inner material ( 37 )
. If the sample is well preserved, bleaching the bone powder can be effective to degrade contaminant DNA preferentially over endogenous DNA
( 40
) .

Ancient DNA has been obtained from many different kinds of substrates. Hair has recently shown excellent promise for the preservation of aDNA and exclusion of environmental contaminants
( 41
) , as was highlighted recently with the publication of the fi rst ancient human genome sequenced from permafrost-preser
ved hair

( 42
) .

However, because of their comparative abundance, bones and teeth remain the most commonly used substrates for aDNA work. When obtaining powder from bones or teeth for extraction (see Note 3), it is important to protect the area where PCR is set up, either by separating or enclosing the bone powdering area or by installing an air vent that will gently draw the bone powder out of the air and separately enclosing the PCR setup area in a fl ow hood or glove box.

The most appropriate protocol for long-term storage of ancient specimens varies depending on how the species were collected. If a sample was frozen upon collection, it is ideal to maintain that temperature. If a sample was collected at room temperature, it should be stored in a cool, dry environment but may not benefi t from being frozen, in particular if several freeze/thaw cycles are anticipated. In general, simply avoiding environmental conditions that are known to promote DNA damage is key to sample preservation.

A cool, dry, temperature-stable environment is ideal. Avoid heat, freeze/thaw cycles, and moisture. Although the depositional and preservation conditions are most important in the survival of DNA through time, poor treatment after sample collection can quickly degrade DNA that has persisted over thousands of years.

Work with aDNA is time-consuming and expensive. However,

when care and appropriate precautions are taken from the outset, it can be a powerful tool for investigating evolutionary processes that cannot be addressed using modern data alone.

4. Notes

1. This also applies to notebooks, carrier bags, jackets,
etc.
It is practical to have a storage area outside the clean lab where personnel can leave their “PCR-contaminated” belongings.

10

T.L. Fulton

2. Carrier DNA should be selected to be a type of DNA that will not be amplifi ed in the PCR reaction. For example, lambda or vector DNA is easily accessible and makes good carrier DNA for mammalian studies.

3. A Dremel rotary tool with a cutting blade is useful for scraping off the surface of a bone, as well as for cutting out sections that can later be powdered using a bone mill. Other bits, such as a Dremel engraving cutter or a regular drill bit, are useful for hollowing out sections of bone underneath the surface, reducing the amount of damage that is visible as well as accessing the inner material that is less likely be contaminated or exposed to damaging agents.

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