TITLE: Multi-Step Ancient DNA Extraction Protocol For Bone And Teeth
AUTHORS: Valeria Mattiangeli, cassidl, Kevin Daly, mullinv, Marta Verdugo

[DESCRIPTION]
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]
SECTION: Stage 1 - Preparation (day 1)
1. In advance of extraction, prepare bone powder. Measure 0.09 mg to 0.120 mg of bone powder into a 2 mL  Eppendorf tube

SECTION: Stage 1 - Preparation (day 1)
2. 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 30 min  (30 minutes) of UV light

 




SECTION: Stage 2 - Extraction (day 1)
3. To each tube add 990 µL of extraction buffer to each tube of bone powder

SECTION: Stage 2 - Extraction (day 1)
4. Cover each tube in parafilm and vortex until the bone pellet is completely in solution.
SECTION: Stage 2 - Extraction (day 1)
5. Incubate   using a thermomixer  .
SECTION: Stage 3 - Preparation (day 2)
6. 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 30 min  (30 minutes) of UV light

 

SECTION: Stage 3 - Preparation (day 2)
7. Remove each tube from incubation, then spin each sample tube using a bench centrifuge for 10 min (10min) at  10000 rpm.
SECTION: Stage 3 - Preparation (day 2)
8. 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"
SECTION: Stage 4 - Extraction (day 2)
9. Add 990 µL of extraction buffer to each sample tube containing the remaining bone pellet.
SECTION: Stage 4 - Extraction (day 2)
10. Cover each tube in parafilm and vortex until the bone pellet is completely in solution.
SECTION: Stage 4 - Extraction (day 2)
11. Incubate   using a thermomixer  .
SECTION: Stage 5 - Preparation (day 3)
13. 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 30 min  (30 minutes) of UV light


 

SECTION: Stage 5 - Preparation (day 3)
12. Remove each tube from incubation, then spin each sample tube using a bench centrifuge for 10 min (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.

SECTION: Stage 5 - Preparation (day 3)
14. 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"
SECTION: Stage 6 (optional) - Extraction (day 3)
15. To each tube add 990 µL of extraction buffer to each tube of bone powder.
SECTION: Stage 6 (optional) - Extraction (day 3)
16. Cover each tube in parafilm and vortex until the bone pellet is completely in solution.
SECTION: Stage 6 (optional) - Extraction (day 3)
17. Incubate   using a thermomixer  .
SECTION: Stage 7 - Purification (final day)
18. Optional: If a third overnight extraction was performed, remove each tube from incubation, then spin each sample tube using a bench centrifuge for 10 min (10 min) at maximum speed  13300 rpm / 17000 x g.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         
SECTION: Stage 7 - Purification (final day)
19. 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.

SECTION: Stage 7 - Purification (final day)
20. 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. 

SECTION: Stage 7 - Purification (final day)
21. Remove the columns from the centrifuge. Add an additional 3 mL of TrisEDTA (x1) to the filters.

SECTION: Stage 7 - Purification (final day)
22. 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.

SECTION: Stage 7 - Purification (final day)
23. Prepare a    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, 1 min (17000 x g). Discard the flow-through. 

SECTION: Stage 7 - Purification (final day)
24. Add 750 µL  of   to each     column. Spin the MinElute columns for 13300 rpm, 1 min (17000 x g). Discard the flow-through. 

SECTION: Stage 7 - Purification (final day)
25. Dry-spin the   columns for 13300 rpm, 1 min (17000 x g).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             
SECTION: Stage 7 - Purification (final day)
26. Place each column in a fresh 1.5 mL labeled collection Eppendorf tube. These are the final tubes and will contain the purified DNA.
SECTION: Stage 7 - Purification (final day)
28. Using a pipette, add 50 µL of EBT (see material) to the filter of each spin column. Wait 5 min (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.
SECTION: Stage 7 - Purification (final day)
29. Sample tubes containing the purified DNA extract should be stored in a -20 °C freezer.
SECTION: Stage 7 - Purification (final day)
27. Heat EBT (50 µL  x n, where n is the number of tubes plus one) to 65 °C using a thermomixer.