Ancient DNA: Methods and Protocols (19 page)

concentration affects the effi cacy of DNA release. As other buffers have also yielded DNA using this method, it may be worthwhile to test the lower GuSCN concentration, especially when working with fragile specimens. Alternatively, after

removal of the sample, you may want to dilute the extraction buffer to 1.5 M GuSCN using TE before adding silica. If nonchaotropic extraction buffers are used (e.g.
( 29
) ), add washing buffer 1 (this can also serve as binding buffer) in a ratio of 2

volumes extraction buffer to one volume washing buffer 1 after removal of the specimen and proceed from step 2 (see also

Note 4).

4. The volume of extraction buffer needs to be adjusted depending on sample size. Tubes or dishes should be large enough to allow samples to move freely within them. Ideally, buffer

should fl ow over the specimen during the agitation; avoid using too little buffer or fi lling up the tubes completely. If using more than 10 mL of extraction buffer, adjust the volume of silica suspension used for DNA binding. Volumes above

50 mL have to be purifi ed in several parallel tubes. When using nonchaotropic salts, it is possible to concentrate the extraction buffer before silica purifi cation using fi lter systems like the Vivafl
ow system

( 32 )
. However, note that chaotropic salts destroy the fi lter membranes. If using nonchaotropic extractions buffers in combination with silica purifi cation, adjust the volume of the GuSCN buffer (washing buffer 1) added for

binding so that the ratio of extraction to binding buffer is 2:1

(
( 31
) and Chap. 3 ; see also Note 6).

5. Rotation during incubation should be gentle so as to avoid damaging fragile specimens. It is also possible to slowly tumble the dishes containing the specimens. Independently of how

98

M. Hofreiter

agitation is achieved, the buffer should fl ow over the specimen in order to get DNA into solution. With some buffers, longer incubation times (i.e., 5–7 days) seem to be benefi cial
( 27 )
, but with GuSCN buffer, a period of 1–2 days is in most cases suffi cient
( 27 )
. With specimens such as arthropods, incubation times of a few hours have been shown to be suffi cient
( 28
) . If contamination of samples with DNA from other species (e.g., human) or other individuals of the same species (cross-contamination) is a problem, it may be benefi cial to incubate samples overnight, discard (or store for potential later uses) the extraction buffer, and then incubate the samples again, and only use the extraction buffer from the second round of incubation for further processing (see Chap. 14 ).

6. The volume of silica suspension has to be adjusted proportionally when different extraction/binding buffer volumes are used. The volume of silica suspension used should be at least 50 m L, as smaller silica volumes yield less DNA. If very large volumes of extraction buffer are used, do not exceed 400 m L of silica suspension per extraction, as it becomes diffi cult to recover all of the TE used for elution. It is possible to increase the elution volume, but if more than 50 mL of extraction/

binding buffer are used, purifi cation in multiple tubes is required, although this will result in higher volumes of extract and thereby less concentrated DNA. Alternatively, when using nonchaotropic extraction buffers, it is possible to concentrate the extraction buffer prior to the adsorption step using appropriate fi lter systems (e.g.
( 32
) , see also Note 2).

7. Vortex silica until it is a homogenous suspension immediately before adding it to the extraction/binding buffer; note that silica particles settle relatively quickly.

8. If possible, keep the supernatant until satisfying results are obtained. If none of the samples yielded amplifi able DNA, it is possible to repeat the silica purifi cation steps by adding freshly made silica suspension and continue from the 3-h incubation step.

9. When using the GuSCN buffer described here, a column

method for washing the silica and DNA elution can be used

(
( 31
) , see also Chap.

3 ) instead of a silica-batch extraction

method. Although the effi ciency of the two methods with this protocol has not been evaluated, given that they perform similarly in ancient DNA extraction
( 31 )
, it is unlikely that any signifi cant differences should occur when combined with the preceding steps of this protocol. It is also possible to use ethanol or isopropanol precipitation in combination with the GuSCN
( 26
) or a modifi ed
( 28 )
buffer. It should be noted that, when a nonchaotropic extraction buffer is used in combination with silica extraction, it is necessary for binding of DNA to the 13 Nondestructive DNA Extraction from Museum Specimens

99

silica to add a chaotropic binding buffer. In our experience, GuSCN in the concentration and volume ratio described in

( 31
) gives the best results with regard to DNA yield and absence of PCR inhibitors.

10. If the silica is still colored after two washing steps, repeat the procedure from step 3. Washing buffer 1 normally reduces the amount of potentially inhibiting coextracted contaminants.

11. It is crucial that washing buffer 2 is removed as completely as possible at this step, as remaining traces of GuSCN can result in incomplete elution of DNA. A second elution will recover most of the remaining DNA, but this may result in lower concentrations of DNA in the fi nal extract if pooled with the fi rst elution (see also Note 6).

12. If more than 100 m L of silica are used for purifi cation, it is recommended to increase the volume of the elution buffer,

although the exact amount has yet to be determined experimentally. The volume of recovered elution buffer should be at least 50 m L (see also Note 6).

13. Low retention or siliconized tubes are recommended for DNA storage, as they reduce DNA loss due to tube wall effects.

Acknowledgments

I thank Beth Shapiro for pestering me until this chapter was written and the University of York for fi nancial support.

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Case Study: Using a Nondestructive DNA Extraction

Method to Generate mtDNA Sequences from Historical

Chimpanzee Specimens*

Elmira Mohandesan , Stefan Prost , and Michael Hofreiter Abstract

A major challenge for ancient DNA (aDNA) studies using museum specimens is that sampling procedures usually involve at least the partial destruction of each specimen used, such as the removal of skin, pieces of bone, or a tooth. Recently, a nondestructive DNA extraction method was developed for the extraction of amplifi able DNA fragments from museum specimens without appreciable damage to the specimen. Here, we examine the utility of this method by attempting DNA extractions from historic (older than 70 years) chimpanzee specimens. Using this method, we PCR-amplifi ed part of the mitochondrial HVR-I region from 65% (56/86) of the specimens from which we attempted DNA extraction. However, we found a high incidence of multiple sequences in individual samples, suggesting substantial cross-contamination among samples, most likely originating from storage and handling in the museums. Consequently, reproducible sequences could be reconstructed from only 79% (44/56) of the successfully extracted samples, even after multiple extractions and amplifi cations. This resulted in an overall success rate of just over half (44/86 of samples, or 51% success), from which 39 distinct HVR-I haplotypes were recovered. We found a high incidence of C to T changes, arguing for both low concentrations of and substantial damage to the endogenous DNA. This chapter highlights both the potential and the limitations of nondestructive DNA extraction from museum specimens.