Ancient DNA: Methods and Protocols (15 page)

Read Ancient DNA: Methods and Protocols Online

Authors: Beth Shapiro

Extraction techniques that incorporate
N
-phenacylthiazolium bromide (PTB) may be appropriate when CTAB and other standard 10 Ancient DNA Extraction from Plants

protocols are unsuccessful. PTB is believed to increase yield by releasing DNA from DNA–protein crosslinks
( 29, 30 )
. PTB has been used in coprolite DNA extractions to counteract the inhibitory effects of Maillard products, and also in extractions of various modern and ancient plant tissues
( 3, 4, 29,
31, 32
) . PTB-based extraction performs very well in modern wood extraction when compared with CTAB and unmodifi
ed Qiagen kit extractions

( 32
) . PTB

extraction has been used to isolate DNA from desiccated maize
cobs without attached seeds ( 3 )
and from modern and ancient bottle gourd rind tissue
( 4
) .

PTB extraction protocols for use with plants have been

described previously
( 3, 32
) and typically include a long incubation period in EDTA, followed by the addition of PTB and often proteinase K, phenol-chloroform extraction, and DNA recovery through pr
ecipitation or silica-binding. Erickson and others ( 4
) developed a simple PTB-based protocol for use with bottle gourd rind. I provide a modifi ed version of this protocol below, in which tissue disruption is followed by an overnight incubation in extraction buffer. The tissue is then centrifuged out of the mixture and DNA is extracted from the supernatant using a Qiagen DNEasy Plant Mini Kit (Qiagen).

2. Materials

2.1. CTAB Protocol

1. CTAB buffer: 2% (w/v) CTAB, 100 mM Tris–HCl pH 8.0,

20 mM EDTA, 1.4 M NaCl. 500 μ L per sample.

2. β -Mercaptoethanol, 5 μ L/1 mL of CTAB buffer.

3. Polyvinylpyrrolidone (PVP), 40 mg/1 mL of CTAB buffer.

4. Chloroform, 500 μ L per sample.

5. Isopropanol, approximately 200–300 μ L per sample, on ice.

6. 7.5 M Ammonium acetate, approximately 25–35

μ L per

sample, on ice.

7. 70% Ethanol, 700 μ L per sample.

8. 95% Ethanol, 700 μ L per sample.

9. TE buffer: 10 mM Tris–HCl pH 8.0, 1 mM EDTA. 50 μ L per sample, more to dilute DNA if necessary.

10. Sterile pellet pestles.

11. 1.5-mL microcentrifuge tubes.

12. Water bath or heat block.

13. Table top centrifuge for 1.5/2-mL tubes capable of

13,000 ×
g
RCF.

14. Fume hood rated for use with chloroform and β -mercaptoethanol.

L. Kistler

2.2. PTB Protocol

1. PTB extraction buffer: 1% SDS, 10 mM Tris, pH 8.0, 5 mM

NaCl. 50 mM DTT, 0.4 mg/mL proteinase K, 10 mM EDTA,

2.5 mM
N
-phenacylthiazolium bromide (PTB). 1.2 mL per sample.

2. Qiagen Plant DNEasy Mini Kits, 1 per sample. The lysis buffer (AP1) and RNaseA included with the kit are not necessary.

3. Shaker bath, or another means of incubating samples at 37°C

with constant agitation.

4. Mechanized mill, such as a bead mill, or a sander wheel attachment on a power drill.

5. 1.5-and 2-mL microcentrifuge tubes.

6. Table top centrifuge for 1.5/2-mL tubes capable of 20,000 ×
g (see Note 1).

3. Methods

3.1. CTAB Protocol

See Notes 2–4 before beginning.

1. Preheat the water bath or heat block to 55°C.

2. Mix the extraction buffer by adding to the CTAB buffer: PVP

to 1 mM (40 mg/mL) and β -mercaptoethanol to 0.5% (v/v,

5mL/mL) (see Notes 5–6). Mix gently by inverting and heat

slightly to dissolve PVP if necessary. Do not shake the buffer, as the detergent will foam easily.

3. Soak one seed in 500 μ L extraction buffer in a 1.5-mL tube for 1 h at 55°C, agitating periodically (see Notes 7–9).

4. Following the incubation period, grid the seed without removing it from the tube using a sterile pellet pestle, and vortex briefl y to homogenize the mixture. No large clumps of tissue should remain (see Note 10).

5. Add 500 μ L of chloroform to each tube and mix gently (see Note 11).

6. Centrifuge for 7 min (see Note 12).

7. Transfer the aqueous phase (top layer) from each tube into a new tube, taking care to leave behind the bottom layer and the debris-fi lled interface.

8. Estimate the volume of the transferred aqueous phase. Add 0.08 volumes of cold ammonium acetate and 0.54 volumes of

cold isopropanol. Invert 20–30 times to mix (see Note 13).

9. Incubate the tubes on ice for at least 30 min and up to 1 h.

10. Centrifuge for 3 min.

11. Carefully discard the supernatant without disturbing the DNA pellet (if visible) (see Note 14).

10 Ancient DNA Extraction from Plants

12. Add 700 μ L of 70% ethanol to each tube, invert 5–10 times, and centrifuge for 1 min.

13. Carefully discard the supernatant without disturbing the DNA pellet (if visible).

14. Add 700 μ L of 95% ethanol, invert 5–10 times, and centrifuge for 1 min.

15. Carefully discard the supernatant without disturbing the DNA pellet (if visible).

16. Invert the tubes on a paper towel briefl y (2–3 min) to eliminate most of the moisture. Then leave the tubes right side up, but covered with a clean paper towel or tissue, until thoroughly dried, at least 1 h or up to overnight.

17. Rehydrate the samples with 50 μ L TE Buffer at room temperature overnight (see Note 15).

3.2. PTB Protocol

See Note 16 before beginning.

1. Preheat the shaker bath (or alternative) to 37°C.

2. Prepare the rind tissue by removing exposed tissue with a sterile razor blade or scalpel. Do not remove the tough outer rind (exocarp), as it tends to yield more DNA than the cork-like inner rind (mesocarp). Wipe the outer rind clean and lightly bleach it, taking care to thoroughly remove all bleach with ethanol and dry the surface completely before extraction.

3. Grind 0.1–0.2 g of rind tissue to a fi ne powder using a mechanized mill or sander wheel, sterilizing the grinding equipment thoroughly between uses (see Note 17).

4. Add the powder to 1.2 mL of PTB extraction buffer in a 2-mL

or larger tube, and vortex to homogenize thoroughly. The

mixture should be somewhat fl uid, not a dry cake in the tube.

Add more PTB extraction buffer, if necessary, to achieve the desired consistency.

5. Incubate the mixture at 37°C with constant agitation for 18–24 h.

6. Centrifuge the mixture at 9,000 ×
g
for 5 min. The samples should separate into a dense mass of tissue and about 500–700 μ L of supernatant. If the tissue is not suitably compacted (i.e. if more than a very small amount of visible debris is suspended in the supernatant), centrifuge for an additional 2 min at up to 16,000 ×
g
.

7. Transfer the supernatant from each tube to a new 1.5-or 2-mL

tube, and estimate the recovered volume.

8. Add 0.325 volumes of Qiagen Buffer AP2, mix, and incubate on ice 5 min.

L. Kistler

9. Complete the extraction by following the manufacturer’s protocol provided with the Qiagen kit, beginning with step 10

(see Notes 18–20).

4. Notes

1. 20,000 ×
g
is recommended in the manufacturer’s protocol for the Qiagen kits, but 13,000 ×
g
is generally adequate.

2. This protocol was developed for chenopods, which are weedy dicots that produce small (1–2 mm diameter), starchy seeds.

When working with monocots, note that monocot seeds store

nutrients for the embryo in starchy endosperm that often comprises the bulk of the seed tissue (e.g. cereals), while dicots use large cotyledons for storage. Cotyledons form with normal

ploidy and become crucial photosynthetic organs following

germination, while monocot endosperm in seeds is strictly

used for embryonic nourishment and forms at half the plant’s normal ploidy (e.g. hexaploid breadwheat forms a large amount of triploid endosperm). Relative quantity and placement of plastids might also be important if cpDNA is being targeted.

3. Severe PCR inhibition is sometimes observed in specimens with a heavily lignifi ed epidermal layer. Removal of this tissue prior to extraction increases PCR success dramatically. This type of protective maternal tissue can be removed with a sterile razor blade or scalpel to facilitate DNA extraction directly from the embryo, especially when PCR inhibition is observed.

4. This protocol is not effective with protein-rich, oily bottle gourd (
Lagenaria siceraria
(Molina) Standl.) seeds, even when paired with extra phenol-chloroform purifi cation steps. Similar seeds such as sunfl ower and squash may also be unsuitable.

5. When these components have been added, the shelf life of the buffer is limited (2–3 days), so make only enough for immediate use.

6. All steps using β -mercaptoethanol or chloroform should be performed under a fume hood.

7. To use tissues other than seeds, grind 10–20 mg of desiccated material prior to incubation.

8. To increase yield using larger volumes of tissue, increase the amount of extraction buffer, taking care to use enough so that the plant tissue does not form a semi-solid cake in the tube.

Increase all other reagents prior to TE rehydration proportionally. It may also be necessary to increase tube size.

10 Ancient DNA Extraction from Plants

9. An optional addition of 1.5 μ L RNase A and 15-min incubation at 37°C can be included after step 4, but is typically not necessary when working with ancient samples.

10. For tough samples, add a small amount of sterile sand to assist with grinding. This may not be suffi cient for very tough seeds.

Grind very tough samples using a small mortar and pestle or a mechanized mill prior to incubation, taking care to thoroughly sterilize the equipment using a strong bleach solution (10%) between samples. For small seeds, try to avoid grinding outside the incubation tube to reduce loss of tissue. Degraded ancient samples might be fragile enough for grinding before step 3.

The incubation in buffer helps slightly soften tough samples.

11. Pure chloroform or chloroform:isoamyl alcohol 24:1 may be used.

12. All centrifugation steps should be carried out at

13,000–16,000 ×
g
.

13. The aqueous phase following chloroform extraction is typically 300–350 μ L for small seeds.

14. It is very unlikely that small seeds, especially of ancient origin, will yield a visible DNA pellet.

15. DNA concentration may vary considerably and depends on taxon, tissue type, and sample preservation. Template volume in subsequent PCR and other applications should be optimized accordingly.

16. This protocol is highly effective with bottle gourd (
Lagenaria siceraria
(Molina) Standl.) rind tissue, yielding nuclear and chloroplast DNA that amplifi es easily
( 4 )
. It is recommended for similar tissues, but has not been shown to be effective with wood DNA extraction. DNA yields from wood are expected

to be low, regardless of the extraction protocol used. Given that the secondary xylem undergoes programmed cell death,

including the dissolution of organelles and nucleic acids, the only recoverable DNA in the woody matrix is likely found in the axial and radial parenchyma cells. To use PTB with wood samples, consider using the protocol described by Asif and Cannon
( 32
) . Alternatively, Qiagen kit modifi cations for wood DNA extractions are described elsewhere
( 16– 18, 33
) . CTAB

extraction is not recommended for wood samples.

17. To avoid overheating while grinding with a dremel or drill, grind slowly and monitor temperature closely. If heat becomes problematic, periodically soak the attachment in ice water and dry thoroughly before continuing.

18. A 1.5-mL tube will only accommodate 600 μ L of lysate going into the kit’s step 13. Use a larger tube if more lysate is recovered.

L. Kistler

19. Several repetitions of the kit’s step 14 may be necessary, depending on the volume of lysate recovered in step 12.

20. It is recommended to elute using a total of 100 μ L of Qiagen Buffer AE to increase template concentration. Elution volume may be altered according to sample size and quality.

References

1. Schlumbaum A, Tensen M, Jaenicke-Despres 13. Szabó Z et al (2005) Genetic variation of V (2008) Ancient plant DNA in archaeobot—

melon (

C. melo
) compared to an extinct

any. Veg Hist Archaeobot 17(2):233–244

landrace from the Middle Ages (Hungary). I.

2. Gugerli F, Parducci L, Petit RJ (2005) Ancient

rDNA, SSR and SNP analysis of 47 cultivars.

plant DNA: review and prospects. New Phytol

Euphytica 146:87–94

166:409–418

14. Kistler L, Shapiro B (2011) Ancient DNA con—