Ancient DNA: Methods and Protocols (14 page)

3. Qiagen Kit containing: Qiagen columns, PBi buffer (see Note 5), and EB buffer.

4. AW1 wash buffer.

5. AW2 wash buffer.

6. Table top centrifuge for 1.5-and 2.0-mL tubes capable of approximately 16,000 ´
g
).

9 DNA Extraction from Fossil Eggshell

67

3. Methods

Procedures should be carried out at room temperature unless otherwise specifi ed. All surfaces and equipment should be cleaned with bleach and then ethanol to eliminate contamination. Always include extraction negative controls.

3.1. Eggshell Sampling

1. Prior to sampling, using either dremel tool (#114 or #191 drill bit) or sandpaper, lightly grind off the outer surfaces of the eggshell sample to remove debris (see Note 1).

2. Working on top of a clean piece of clean aluminium foil and using a clean drill bit, powder 50–100 mg of eggshell (see Note 2).

3. Transfer the eggshell powder from the foil to a pre-weighed 2.0-mL safelock tube for digestion. Weigh the tube to determine the exact amount of powder used in the DNA isolation procedure.

3.2. Eggshell Digestion

1. Add 7 m L DTT solution and 14 m L proteinase K solution per 679 m L stable digestion buffer (EDTA, Tris, Triton X-100) to make the active digestion buffer (see Note 3). Mix well. Seal with parafi lm and place in a rotating oven (or thermal mixer) for 5 min at 55°C to dissolve Triton X-100 (see Note 4).

2. Add the active digestion buffer (700 m L) to a 2.0 mL Eppendorf containing 50–100 mg of eggshell powder and seal with para-fi lm (a good seal is essential). Gently vortex the tube to homogenize the digestion buffer with the eggshell powder. Incubate with rotation for 2–24 h at 55°C.

3. Increase the temperature of the oven or block to 95°C.

Meanwhile, vortex the sample tubes for 20 s. Once the desired temperature has been reached, incubate samples for 10 min at 95°C. Vortex each sample and repeat the heating step again.

Or repeat step 3. (see Note 6).

4. Allow tubes to cool to room temperature on a bench and remove parafi lm.

5. Proceed to DNA purifi cation (below).

3.3. DNA Purifi cation:

1. Following digestion, centrifuge the sample at 16,000 ´
g
for 2 min
Silica Method

and ensure any remaining undigested eggshell has settled.

2. Collect the supernatant and transfer to a 30,000 MWCO

Vivaspin 500 column (Sartorius Stedim Biotech, Germany; see Note 7).

3. Centrifuge the Vivaspin column with the supernatant at 16,000 ´
g
for 10–20 min, to concentrate supernatant to ~50 m L.

68

C.L. Oskam and M. Bunce

4. Transfer the concentrated supernatant to a new 2-mL tube and combine with at least 5 volumes of Qiagen Buffer PBi and

vortex to mix (see Note 5
)
.

5. Using a bench centrifuge, spin sample for 10 s and add to a Qiaquick column with attached collection tube.

6. Centrifuge Qiaquick column for 1 min at 16,000 ´
g
, and discard the fl ow-through.

7. Wash with 700 m L Qiagen wash buffer AW1 by centrifuging sample for 1 min, and discard the fl ow-through.

8. Wash with 700 m L Qiagen wash buffer AW2 by centrifuging sample for 1 min, and discard the fl ow-through.

9. To ensure all buffer components have been removed, centrifuge for an additional 1 min. Then place the Qiaquick column in a clean 1.5-mL tube with the lid removed.

10. To elute the DNA, add 60 m L (or a volume appropriate to the concentration of DNA required) of Qiagen elution buffer EB

directly to the centre of the silica membrane. Wait 5 min prior to centrifugation to allow the DNA to elute off the silica.

11. Centrifuge for 1 min at maximum speed to collect the EB, now containing DNA.

12. Transfer to new 1.5-mL tubes (that have lids). The DNA is ready for downstream molecular biology analyses (see

Note 8).

4. Notes

1. Thin eggshell is very fragile and susceptible to crumbling. We recommend using a dremel with drill bit for thicker (>0.7 mm) and sandpaper for thinner eggshell fragments (<0.7 mm) to remove the outer surfaces of the eggshell.

2. Following the removal of the outer surfaces, thin eggshell becomes more fragile. To ensure a homogenous powder, a

mortar and pestle may be preferred as using a dremel may

result in ‘chipped off’ pieces of thin eggshell.

3. Make up fresh for each digestion and discard unused solution.

DTT and proteinase K are not stable when added to make an

active digestion solution; it is for that reason that the active buffer is made fresh for each digestion.

4. Detergents (i.e. Triton X-100) at 4°C will precipitate out of solution in the digestion buffer. If this occurs, the digest buffer should be heated to allow the detergent to fully dissolve. SDS

(1%) can be substituted for 1% Triton X-100; however, an

9 DNA Extraction from Fossil Eggshell

69

increased level of inhibition has been observed during qPCR

experiments (see
( 5
) ).

5. We have noticed that eggshells that have been thermally modifi ed (i.e. burnt archaeological eggshell) may alter the pH during the DNA-binding step to the silica. This is observed as a change in pH colour, from a preferred yellow (pH <7.5) to a light–dark purple (pH >7.5), therefore it is imperative that the pH indicator (now supplied separately) is added to the Qiagen buffer PB before use. The pH change can be overcome by the addition of ~2 m L of 3 M sodium acetate (pH 5.2) to the 5

volumes of Qiagen buffer PBi. As it may take a few seconds to see a colour change, be sure to mix thoroughly before adding additional sodium acetate.

6. Although not all of the powdered eggshell (calcium carbonate) will digest, this 95°C heat step is particularly important, as we believe the heat releases the DNA from the intracrystalline eggshell matrix. However, it should be noted that disruption of the DNA duplex at 95°C may cause problems with downstream applications that require adapter ligation such as HTS

library builds.

7. The 30,000 MWCO Vivaspin 500 columns in this protocol serve two purposes. First, small molecules that act as PCR

inhibitors are allowed to pass through the column while the DNA is retained. Second, the MWCO membrane acts to concentrate the DNA into a volume more appropriate for the silica-binding step. The vertical polyethersulfone (PES) membranes of the Vivaspin 500 column (Sartorius Stedim

Biotech, Germany) are preferable to those that use horizontal membranes (often cellulose-based), as they are more resistant to blockage. Whatever MWCO membrane is used, large differences in the fl ow rate of different samples through the columns are still commonly observed.

8. Investigators should be cognisant that it is not uncommon for the DNA extract to contain inhibitors detrimental to PCR;

therefore, we recommend that a dilution series using qPCR is performed directly after the extraction of DNA to assess any inhibition and the best level of dilution for further use of the extract.

Acknowledgments

MB was supported by the Australian Research Council as a Future Fellow (FT0991741). We thank Emma McLay, Morten Allentoft, Jayne Houston, and James Haile for helpful advice.

70

C.L. Oskam and M. Bunce

References

1.

Higham T (1994) Radiocarbon dating New

epimerization in ostrich eggshell. Quat Sci Rev

Zealand prehistory with moa eggshell: some

18:1537–1548

preliminary results. Quat Sci Rev 13:163–169

4.

Miller GH, Fogel ML, Magee JW, Gagan MK,

2.

Johnson BJ, Miller GH, Fogel ML, Beaumont

Clarke SJ, Johnson BJ (2005) Ecosystem col—

PB (1997) The determination of the late

lapse in Pleistocene Australia and a human role

Quaternary paleoenvironments at Equus Cave,

in megafauna extinction. Science 309:287–290

South Africa, using stable isotopes and amino 5. Oskam CL, Haile J, McLay E, Rigby P, Allentoft acid racemization in ostrich eggshell.

ME, Olsen ME, Bengtsson C, Miller GH,

Palaeogeogr Palaeoclimatol Palaeoecol Schwenninger JL, Jacomb C, Walter R, Baynes 136:121–137

A, Dortch J, Parker-Pearson M, Gilbert MT,

3.

Miller GH, Beaumont PB, Deacon HJ, Brooks

Holdaway RN, Willerslev E, Bunce M (2010)

AS, Hare PE, Jull AJT (1999) Earliest modern

Fossil avian eggshell preserves ancient DNA.

humans in southern Africa dated by isoleucine

Proc Biol Sci 277:1991–2000

Logan Kistler

Abstract

A variety of protocols for DNA extraction from archaeological and paleobotanical plant specimens have been proposed. This is not surprising given the range of taxa and tissue types that may be preserved and the variety of conditions in which that preservation may take place. Commercially available DNA extraction kits can be used to recover ancient plant DNA, but modifi cations to standard approaches are often necessary to improve yield. In this chapter, I describe two protocols for extracting DNA from small amounts of ancient plant tissue. The CTAB protocol, which I recommend for use with single seeds, utilizes an incubation period in extraction buffer and subsequent chloroform extraction followed by DNA purifi -

cation and suspension. The PTB protocol, which I recommend for use with gourd rind and similar tissues, utilizes an overnight incubation of pulverized tissue in extraction buffer, removal of the tissue by centrifugation, and DNA extraction from the buffer using commercial plant DNA extraction kits.

Key words:
Ancient DNA , Plant DNA , DNA extraction , CTAB extraction , PTB extraction 1. Introduction

DNA recovered from archaeological and paleobotanical plant remains can be used to infer plant domestication histories, the movement of crop plants, paleoecological community models, and plant demographic histories
( 1– 6 )
. Plant tissues preserved by desiccation or freezing are ideal for ancient DNA (aDNA) research, but even samples that have been subjected to anaerobic waterlogging and carbonization have been shown to contain amplifi able DNA
( 7– 9 )
.

There is currently no standard protocol for DNA extraction from ancient plant remains, due largely to the diversity of plant taxa and tissue types recovered in ancient deposits. Leaf tissue is a preferred material for modern DNA extraction, but is rarely available archaeologically. Seeds are often used in aDNA analyses and Beth Shapiro and Michael Hofreiter (eds.),
Ancient DNA: Methods and Protocols
, Methods in Molecular Biology, vol. 840, DOI 10.1007/978-1-61779-516-9_10, © Springer Science+Business Media, LLC 2012

71

72

L. Kistler

have been shown to yield DNA after desiccation, waterlogging, and carbonization
( 7– 14 )
. Seeds preserved by anaerobic soil con-

ditions, while uncommon (e.g. 15
) , might also yield recoverable DNA. Other ancient plants tissues yielding DNA have included gourd rind
( 4
) , maize cobs
( 3
) , wood
( 16– 18 )
, and vegetative tissues such as peatmoss shoots and seagrass rhizomes
( 19, 20
) . Like seeds, pollen grains are adapted for viable DNA storage and protection during dispersal, and pollen grains recovered from lake sediment cores have yielded ancient DNA
( 5, 21 )
. Plant DNA has also been successfully recovered from the inner surfaces of ceramics from an archaeological shipwreck, revealing the contents of ancient Gr
eek amphorae ( 22 )
. DNA isolation has been attempted from siliceous phytoliths extracted from archaeological soils, but has not been successful
( 23
) . Further experimental studies are necessary to understand whether this failure is related to the silicifi cation processes in plants, the archaeological contexts from which phytoliths were taken for aDNA extraction, or the protocols used.

Isolation of DNA from plant tissues is complicated by the presence of abundant polyphenols, sugars, secondary compounds, and other potential PCR inhibitors. Standard DNA extraction protocols and commercial kits designed to overcome these obstacles are often suitable for use with ancient plant remains
( 12, 14, 17– 19,

24, 25 )
. Modifi cations to manufacturers’ protocols are often required, however, to accommodate different tissue types and preservation conditions. Modifi cations range from changes in incubation time and temperature to the use of additional reagents to combat PCR inhibition.

DNA extraction protocols based on the strong detergent cetyl-trimethyl ammonium bromide (CTAB) have been used with plants since the mid-1980s
( 26, 27
) . They can be used with small amounts of tissue, including very small single seeds, and are adaptable to samples of various composition and preservation. CTAB protocols used previously with ancient plant remains, including CTAB/

DTAB variations, are described elsewhere
( 7, 11, 20, 24
) . A protocol developed for modern leaf tissue
( 28 )
and modifi ed for use with single chenopod (
Chenopodium
sp.) seeds ( 14
) is described here. This protocol successfully recovered up to 6 μ g of bulk DNA from single modern seeds, from which PCR products of approximately 1,300 base pairs (bp) amplifi ed easily. The yield was lower and fragment lengths shorter when working with ancient plants, but PCR products amplifi ed successfully from several single-seed extractions. This protocol begins with tissue disruption and incubation in extraction buffer, followed by DNA extraction using chloroform and purifi cation using isopropanol and ammonium acetate. DNA is then washed in ethanol and suspended in TE

Buffer.