Multi-Step Ancient DNA Extraction Protocol For Bone And Teeth
Valeria Mattiangeli, cassidl, Kevin G Daly, Victoria E Mullin, Marta Verdugo
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.
Steps
Stage 1 - Preparation (day 1)
In advance of extraction, prepare bone powder. Measure 0.09mg to 0.120mg of bone powder into a 2mL 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 0h 30m 0s (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 |
Stage 2 - Extraction (day 1)
To each tube add 990µL 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 using a thermomixer 700rpm.
Stage 3 - Preparation (day 2)
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 0h 30m 0s (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 |
Remove each tube from incubation, then spin each sample tube using a bench centrifuge for 0h 10m 0s (10min) at 10000rpm,0h 0m 0s.
Transfer the supernatant to new labelled 1.5mL tubes, parafilm and freeze.
Note: DNA can be purified from this supernatant. Defrost and move onto "Stage 7: Purification"
Stage 4 - Extraction (day 2)
Add 990µL 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 using a thermomixer 700rpm.
Stage 5 - Preparation (day 3)
Remove each tube from incubation, then spin each sample tube using a bench centrifuge for 0h 10m 0s (10 min) at maximum speed 13300rpm,0h 0m 0s / 17000x g,0h 0m 0s.
If a substantial amount of bone pellet remains, an additional extraction step can be performed, for a total of three overnight digestions.
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 0h 30m 0s (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 |
Optional additional extraction: transfer the supernatant to new labelled 1.5mL 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)
To each tube add 990µL 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 using a thermomixer 700rpm.
Stage 7 - Purification (final day)
Optional: If a third overnight extraction was performed, remove each tube from incubation, then spin each sample tube using a bench centrifuge for 0h 10m 0s (10 min) at maximum speed 13300rpm,0h 0m 0s / 17000x g,0h 0m 0s.
Prepare labelled Amicon filter columns, 1 per sample. Add 3mL of TrisEDTA (x1) to each Amicon filter and add the supernatant (~1mL ) 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 2500rpm,0h 0m 0s (1200x g,0h 0m 0s ) in a centrifuge with a swing bucket rotor until the supernatant + TrisEDTA is at the 250µL mark . This is typically 6-10 minutes, but sometimes longer.
Remove the columns from the centrifuge. Add an additional 3mL of TrisEDTA (x1) to the filters.
Spin at 2500rpm,0h 0m 0s (1200x g,0h 0m 0s ) in a centrifuge with a swing bucket rotor until the supernatant + TrisEDTA is at the 100µL mark . Discard flow through and keep the remaining 100µL in the filter.
Prepare a 500µL of PB and add the remaining 100µL from the Amicon filter. Spin the MinElute columns for 13300rpm (17000x g,0h 0m 0s). Discard the flow-through.
Add 750µL of 13300rpm (17000x g,0h 0m 0s). Discard the flow-through.
Dry-spin the 13300rpm (17000x g,0h 0m 0s).
Place each column in a fresh 1.5mL labeled collection Eppendorf tube. These are the final tubes and will contain the purified DNA.
Heat EBT (50µL x n , where n is the number of tubes plus one) to 65°C using a thermomixer.
Using a pipette, add 50µL of EBT (see material) to the filter of each spin column. Wait 0h 5m 0s (5 min), and spin down columns at maximum speed 13300rpm,0h 0m 0s / 17000x g,0h 0m 0s. Collection tube lids should be angled away from the direction of spin.
Sample tubes containing the purified DNA extract should be stored in a -20°C freezer.
