May 03, 2023
Multi-Step Ancient DNA Extraction Protocol For Bone And Teeth
- 1Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland;
- 2University College Dublin
Protocol Citation: Valeria Mattiangeli, cassidl, Kevin G Daly, Victoria E Mullin, Marta Verdugo 2023. Multi-Step Ancient DNA Extraction Protocol For Bone And Teeth. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr45b2gmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 29, 2023
Last Modified: May 03, 2023
Protocol Integer ID: 81182
Keywords: DNA, ancient DNA, extraction
Funders Acknowledgement:
European Research Council
Grant ID: 885729-AncestralWeave
Science Foundation Ireland
Grant ID: 21/PATH-S/9515
Abstract
The protocol described here is a multi-day extraction protocol for the recovery of fragment DNA molecules from bone or teeth powder obtained from ancient or historical remains.
The protocol is based on a silica-column method described initially in (Yang et al, 1998). Further modifications were made to this base protocol and reported (MacHugh et al, 2000), (Gamba et al, 2014), (Daly et al, 2018), and (Verdugo et al, 2019).
The instructions presented here describe the totality of these modifications and are one of the aDNA extraction methods employed by the Molecular Population Genetics group at Trinity College Dublin.
Materials
Sodium Hypochlorite (14%, dilute to 0.5%)
Water (Laboratory grade)
EDTA (0.5M, pH 8)
Tris-HCl (1M, pH 7.4)
SDS (2% final)
Proteinase K (2.5 U/mg or 50U/mL)
Tween (20%)
Qiagen PB, PE, and EB Buffers
EBT buffer: 7.5 ul of 20% Tween in 15ml EB buffer
1.5 ml Eppendorf tubes
Parafilm
Thermomixer
Standard bench centrifuge
Protocol materials
MinEluteQiagen
In 3 steps
PE buffer QiagenCatalog #19065
Step 24
Safety warnings
The described protocol should be performed in dedicated ancient DNA facilities. Workers should wear full-body PPE (gloves x2, body suits, masks) to avoid contamination of material. Extra care must be made to avoid cross-contamination of low concentration material typical of ancient/historical DNA projects.
Stage 1 - Preparation (day 1)
Stage 1 - Preparation (day 1)
30m
30m
In advance of extraction, prepare bone powder. Measure 0.09 mg to 0.120 mg of bone powder into a 2 mL Eppendorf tube
Prepare the extraction buffer where n is the number of sample and control tubes plus one (for pipetting error). In the example below 15 sample tubes and one extraction control tube are used (15 + 1 + 1 = 17)
When preparing the extraction buffer and prior to the addition of proteinase K, subject the buffer to 00:30:00 (30 minutes) of UV light
| A | B | C | D | |
| Reagent | x 1 (ul) | x 17 (ul) | x n (ul) | |
| Tris-HCl, 1M | 20 | 340 | ||
| SDS (2% final) | 17 | 289 | ||
| EDTA, 0.5 | 940 | 15,980 | ||
| *** UV Prior To The Addition Of Proteinase K *** | ||||
| Proteinase K (50 U/mL) | 13 | 221 |
30m
Stage 2 - Extraction (day 1)
Stage 2 - Extraction (day 1)
To each tube add 990 µL of extraction buffer to each tube of bone powder
Cover each tube in parafilm and vortex until the bone pellet is completely in solution.
Incubate Overnight (20-24h) using a thermomixer 700 rpm, 37°C .
Stage 3 - Preparation (day 2)
Stage 3 - Preparation (day 2)
40m
40m
Prepare a fresh batch of extraction buffer where n is the number of sample and control tubes plus one (for pipetting error). In the example below 15 sample tubes and one extraction control tube are used (15 + 1 + 1 = 17)
When preparing the extraction buffer and prior to the addition of proteinase K, subject the buffer to 00:30:00 (30 minutes) of UV light
| A | B | C | D | |
| Reagent | x 1 (ul) | x 17 (ul) | x n (ul) | |
| Tris-HCl, 1M | 20 | 340 | ||
| SDS (2% final) | 17 | 289 | ||
| EDTA, 0.5 | 940 | 15,980 | ||
| *** UV Prior To The Addition Of Proteinase K *** | ||||
| Proteinase K (50 U/mL) | 13 | 221 |
30m
Remove each tube from incubation, then spin each sample tube using a bench centrifuge for 00:10:00 (10min) at 10000 rpm .
10m
Transfer the supernatant to new labelled 1.5 mL tubes, parafilm and freeze.
Note: DNA can be purified from this supernatant. Defrost and move onto "Stage 7: Purification"
Stage 4 - Extraction (day 2)
Stage 4 - Extraction (day 2)
Add 990 µL of extraction buffer to each sample tube containing the remaining bone pellet.
Cover each tube in parafilm and vortex until the bone pellet is completely in solution.
Incubate Overnight (20-24h) using a thermomixer 700 rpm, 37°C .
Stage 5 - Preparation (day 3)
Stage 5 - Preparation (day 3)
40m
40m
Remove each tube from incubation, then spin each sample tube using a bench centrifuge for 00:10:00 (10 min) at maximum speed 13300 rpm / 17000 x g .
If a substantial amount of bone pellet remains, an additional extraction step can be performed, for a total of three overnight digestions.
10m
Optional additional extraction: prepare a fresh batch of extraction buffer where n is the number of sample and control tubes plus one (for pipetting error). In the example below 15 sample tubes and one extraction control tube are used (15 + 1 + 1 = 17)
When preparing the extraction buffer and prior to the addition of proteinase K, subject the buffer to 00:30:00 (30 minutes) of UV light
| A | B | C | D | |
| Reagent | x 1 (ul) | x 17 (ul) | x n (ul) | |
| Tris-HCl, 1M | 20 | 340 | ||
| SDS (2% final) | 17 | 289 | ||
| EDTA, 0.5 | 940 | 15,980 | ||
| *** UV Prior To The Addition Of Proteinase K *** | ||||
| Proteinase K (50 U/mL) | 13 | 221 |
30m
Optional additional extraction: transfer the supernatant to new labelled 1.5 mL tubes, parafilm and freeze.
Note: DNA can be purified from this supernatant. Defrost and move onto "Stage 7 - Purification"
Stage 6 (optional) - Extraction (day 3)
Stage 6 (optional) - Extraction (day 3)
To each tube add 990 µL of extraction buffer to each tube of bone powder.
Cover each tube in parafilm and vortex until the bone pellet is completely in solution.
Incubate Overnight (20-24h) using a thermomixer 700 rpm, 37°C .
Stage 7 - Purification (final day)
Stage 7 - Purification (final day)
18m
18m
Optional: If a third overnight extraction was performed, remove each tube from incubation, then spin each sample tube using a bench centrifuge for 00:10:00 (10 min) at maximum speed 13300 rpm / 17000 x g .
10m
Prepare labelled Amicon filter columns, 1 per sample. Add 3 mL of TrisEDTA (x1) to each Amicon filter and add the supernatant (~1 mL ) to the appriopriate column, taking care not to transfer bone powder. The remaining bone powder tubes and can frozen and re-extracted if needed.
Spin columns at 2500 rpm (1200 x g ) in a centrifuge with a swing bucket rotor until the supernatant + TrisEDTA is at the 250 µL mark. This is typically 6-10 minutes, but sometimes longer.
Remove the columns from the centrifuge. Add an additional 3 mL of TrisEDTA (x1) to the filters.
Spin at 2500 rpm (1200 x g ) in a centrifuge with a swing bucket rotor until the supernatant + TrisEDTA is at the 100 µL mark. Discard flow through and keep the remaining 100 µL in the filter.
Prepare a MinEluteQiagen for each sample with 500 µL of PB and add the remaining 100 µL from the Amicon filter. Spin the MinElute columns for 13300 rpm, 00:01:00 (17000 x g ). Discard the flow-through.
1m
Add 750 µL of PE buffer QiagenCatalog #19065 to each MinEluteQiagen column. Spin the MinElute columns for 13300 rpm, 00:01:00 (17000 x g ). Discard the flow-through.
1m
Dry-spin the MinEluteQiagen columns for 13300 rpm, 00:01:00 (17000 x g ).
1m
Place each column in a fresh 1.5 mL labeled collection Eppendorf tube. These are the final tubes and will contain the purified DNA.
Heat EBT (50 µL x n, where n is the number of tubes plus one) to 65 °C using a thermomixer.
Using a pipette, add 50 µL of EBT (see material) to the filter of each spin column. Wait 00:05:00 (5 min), and spin down columns at maximum speed 13300 rpm / 17000 x g . Collection tube lids should be angled away from the direction of spin.
5m
Sample tubes containing the purified DNA extract should be stored in a -20 °C freezer.
Protocol references
Yang, D. Y., Eng, B., Waye, J. S., Dudar, J. C., & Saunders, S. R. (1998). Technical note: improved DNA extraction from ancient bones using silica-based spin columns. American Journal of Physical Anthropology, 105(4), 539–543. https://doi.org/10.1002/(SICI)1096-8644(199804)105:4<539::AID-AJPA10>3.0.CO;2-1
MacHugh, D. E., Edwards, C. J., Bailey, J. F., Bancroft, D. R., & Bradley, D. G. (2000). The extraction and analysis of ancient DNA from bone and teeth: a survey of current methodologies. Ancient Biomolecules, 3(2), 81–103.
Gamba, C., Cristina, G., Jones, E. R., Teasdale, M. D., McLaughlin, R. L., Gloria, G.-F., Valeria, M., László, D., Ivett, K., Ildikó, P., Alexandra, A., Alasdair, W., János, D., Pál, R., Higham, T. F. G., Michael, H., Bradley, D. G., & Ron, P. (2014). Genome flux and stasis in a five millennium transect of European prehistory. Nature Communications, 5, 5257. https://doi.org/10.1038/ncomms6257
Daly, K. G., Maisano Delser, P., Mullin, V. E., Scheu, A., Mattiangeli, V., Teasdale, M. D., Hare, A. J., Burger, J., Verdugo, M. P., Collins, M. J., Kehati, R., Erek, C. M., Bar-Oz, G., Pompanon, F., Cumer, T., Çakırlar, C., Mohaseb, A. F., Decruyenaere, D., Davoudi, H., … Bradley, D. G. (2018). Ancient goat genomes reveal mosaic domestication in the Fertile Crescent. Science, 361(6397), 85–88. https://doi.org/10.1126/science.aas9411
Verdugo, M. P., Mullin, V. E., Scheu, A., Mattiangeli, V., Daly, K. G., Maisano Delser, P., Hare, A. J., Burger, J., Collins, M. J., Kehati, R., Hesse, P., Fulton, D., Sauer, E. W., Mohaseb, F. A., Davoudi, H., Khazaeli, R., Lhuillier, J., Rapin, C., Ebrahimi, S., … Bradley, D. G. (2019). Ancient cattle genomics, origins, and rapid turnover in the Fertile Crescent. Science, 365(6449), 173–176. https://doi.org/10.1126/science.aav1002