Dec 11, 2020
Ancient DNA Extraction from Dental Calculus
Forked from Ancient DNA Extraction from Skeletal Material
- Franziska Aron1,
- Courtney Hofman2,
- Zandra Fagernäs3,
- Irina Velsko4,
- Eirini Skourtanioti4,
- Guido Brandt3,
- Christina Warinner3
- 1Friedrich-Schiller Universität Jena;
- 2Laboratories of Molecular Anthropology and Microbiome Research, University of Oklahoma;
- 3Department of Archaeogenetics, Max Planck Institute for the Science of Human History;
- 4Max Planck Institute for Evolutionary Anthropology
- WarinnerGroup
- MPI EVA Archaeogenetics
Protocol Citation: Franziska Aron, Courtney Hofman, Zandra Fagernäs, Irina Velsko, Eirini Skourtanioti, Guido Brandt, Christina Warinner 2020. Ancient DNA Extraction from Dental Calculus. protocols.io https://dx.doi.org/10.17504/protocols.io.bidyka7w
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: July 09, 2020
Last Modified: December 11, 2020
Protocol Integer ID: 39064
Keywords: Dabney, aDNA, extraction, DNA extraction, nucleic acids, ancient DNA, palaeogenetics, archaeogenetics, paleogenetics, archeogenetics, dental calculus
Abstract
Silica-based total DNA extraction protocol optimised for the recovery of ultra-short DNA molecules from archaeological dental calculus, modified from Dabney et al. (2013) PNAS (doi: 10.1073/pnas.1314445110) and adapted for dental calculus by Mann et al. (2018) Scientific Reports (doi: https://doi.org/10.1038/s41598-018-28091-9).
Image Attribution
Dental calculus deposit buildup on the molar of a Neanderthal; photo courtesy of Julie Arnaud, University of Ferrara
Guidelines
Working in an Ancient DNA Laboratory
- All steps of the protocol should take place in a clean room facility specifically designed for ancient DNA.
- The researcher performing lab work should wear correspondingly suitable lab-wear, such as:
- full-body suit with hood (e.g., Tyvek)
- hairnet
- face mask
- two pairs of clean gloves (do not wear latex gloves if samples will be used for proteomic analyses)
- clean shoes
- protective glasses
- Sample processing should be carried out in separated work benches with integrated UV irradiation (e.g. Dead Air PCR work bench)
- Surfaces and equipment should be regularly decontaminated with e.g. bleach solution or Thermofisher's DNA AWAY (or similar) and irradiated with UV.
- All home-made buffers should be prepared in a separate, dedicated PCR-free ultra-clean room and UV-irradiated for 30 min.
Please see the following for more detailed guidance:
Llamas, B. et al., 2017. From the field to the laboratory: Controlling DNA contamination in human ancient DNA research in the high-throughput sequencing era. STAR: Science & Technology of Archaeological Research, 3(1), pp.1–14. Available at: https://doi.org/10.1080/20548923.2016.1258824.
Definitions
Stock-aliquot refers to a personal 'stock' (e.g. in a 50ml Falcon Tube) of reagents you can use across multiple sessions of this protocol. An 'aliquot' refers to a sub-aliquot of the stock, that is used for a single session of this specific protocol.
Protocol Specific Guidelines
This protocol requires the use of two rooms - a dedicated PCR-free ultra-clean buffer preparation room and a DNA extraction room. As calculus typically contains higher biomass than bones or teeth, it is preferable to do calculus extractions in a separate space when one is available. This reduces the possibility of contaminating extractions from lower biomass samples with oral bacterial DNA.
Materials
MATERIALS
ParafilmBiozymCatalog #743311
pH indicator strips MQuant® Supelco®Merck MilliporeCatalog #1.09535.0001
Safe-Lock Tubes 1.5 ml PCR clean DNA LoBindEppendorfCatalog #0030108051
Safe-Lock Tubes 2 ml PCR clean DNA LoBindEppendorfCatalog #0030108078
50 ml CELLSTAR® Polypropylene Tube 30/115 MM Conical Bottom Blue screw cap sterile skirtgreiner bio-oneCatalog #210261
EDTA (0.5 M) pH 8.0-500 mLThermo Fisher Scientific AustraliaCatalog #AM9261
Ethanol AbsoluteMerck MilliporeCatalog #1009831011
Guanidine hydrochloride for molecular biology >=99%Sigma AldrichCatalog #G3272-500g
2-Propanol for AnalysisMerck MilliporeCatalog #1070222511
Proteinase K from Tritirachium album lyophilized powder >=30 units/mg proteinSigma AldrichCatalog #P6556-100MG
Sodium Acetate buffer solution 3 M pH 52 for molecular biologySigma AldrichCatalog #S7899-500ML
TE buffer (1X) pH 8.0 low EDTA for molecular biology 500mlPanreac AppliChemCatalog #A8569,0500
TWEEN 20 for molecular biology viscous liquidSigma AldrichCatalog #P9416-50ML
Water HPLC PlusSigma AldrichCatalog #34877-2.5L-M
High Pure Viral Nucleic Acid Large Volume KitRocheCatalog #05114403001
Aluminium foil 30cmx20m Schachtel 1 Ro/VECatalog #3760062
Lab Equipment
PCR work bench (e.g. AirClean Dead Air PCR Werkbank, 48´´)
UV irradiation box or cross linker (e.g. Vilber Lourmat Bio-Link BLX-254)
Incubator with natural convection (e.g. Thermo Scientific Heratherm General Protocol Inkubator IGS100)
Overhead tube rotator (e.g. Stuart SB2/SB3 Rotator)
Centrifuge 50 ml (e.g. Thermo Scientific Heraeus Megafuge 8)
Centrifuge Rotor 50 ml (e.g. Thermo Scientific TX-400)
Centrifuge 1.5/2.0 ml (e.g. Eppendorf 5424)
Centrifuge Rotor 1.5/2.0ml (e.g. Eppendorf F-45-24-11)
Balance (e.g. Ohaus Adventurer balance AX1502)
Vortex mixer (e.g. Scientific Industries Vortex-Genie® 2)
Microwave (Optional)
Glass bottle (e.g., 500 ml)
Generic Reagents
Solution of household bleach (2-6% NaClO, then diluted to a working solution concentration of 0.2-0.5% NaClO)
Thermofisher DNA AWAY
Paper towels or tissues
Safety warnings
Reagents
Household bleach solution (2-6%) diluted to a working concentration of 0.2-0.5 % NaClO in total
- H290 May be corrosive to metals.
- H314 Causes severe skin burns and eye damage.
- H411 Toxic to aquatic life with long lasting effects.
- EUH206 Warning! Do not use together with other products. May release dangerous gases (chlorine). Remove from surface after recommended incubation time with water-soaked tissue.
DNA AWAY
- H314 Causes severe skin burns and eye damage.
Note: Both bleach solutions and DNA AWAY are used for decontamintation. DNA AWAY is less corrosive than bleach and should be preferred for decontamination of sensitive equipments such as surfaces of electric devices.
GuHCl
- H302 Harmful if swallowed.
- H332 Harmful if inhaled.
- H315 Causes skin irritation.
- H319 Causes serious eye irritation.
Ethanol
- H225 Highly flammable liquid and vapour.
- H319 Causes serious eye irritation.
Isopropanol
- H225 Highly flammable liquid and vapour.
- H319 Causes serious eye irritation.
- H336 May cause drowsiness or dizziness.
EDTA
- H373 May cause damage to organs through prolonged or repeated exposure.
Proteinase K
- H315 Causes skin irritation.
- H319 Causes serious eye irritation.
- H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled.
- H335 May cause respiratory irritation.
Sodium Acetate
- H139: Causes serious eye irritation
Kits
Check manufacturer's safety information for the High Pure Viral Nucleic Acid Large Volume Kit used in this protocol.
Equipment
UV radiation
- UV radiation can damage eyes and can be carcinogenic in contact with skin. Do not look directly at unshielded UV radiation. Do not expose unprotected skin to UV radiation.
- UV emitters generate ozone during operation. Use only in ventilated rooms.
Before start
Planning
This protocol takes four days, out of which three include laboratory work and one is an incubation period.
Only the extraction buffer can be prepared within the DNA extraction room. All other home-made buffers must be prepared in a separate dedicated PCR-free ultra-clean room, and we typically UV-irradiate these for 30 min. Purchased kits should be DNA-free.
Check waste disposal guidance for all reagents in this protocol against your corresponding laboratory regulations.
Equipment
Make sure all necessary equipment is available (see Materials).
Abbreviations
EDTA = Ethylenediaminetetraacetic acid
GuHCl = Guanidinium chloride or Guanidine hydrochloride
HPLC = High Performance Liquid Chromatography (-Grade Water)
NaClO = Sodium hypochlorite
TE = Tris-EDTA
TET = Tris-EDTA-Tween (-buffer)
UV = Ultraviolet (radiation)
Samples
Ensure sample aliquots (2-5mg) of dental calculus (in 2ml tubes) are prepared in a dedicated sampling room, prior to the day you start this protocol.
Controls
Take along a positive control (sample of known performance) and a negative control (empty tube) in order to assess the performance of the protocol and the level of background contamination. Consider these two extra samples in your calculations for buffer preparations.
Day 1: Preparation of reagents (Buffer Prep Room)
Day 1: Preparation of reagents (Buffer Prep Room)
Prepare cleaned workspace with all necessary reagents and equipment.
Note
If lab-wide large-batch pre-prepared reagent stores are used, ensure to have made personal stock-aliquot of reagents such as UV-Water, EDTA, sodium acetate, and proteinase K in amounts sufficient for this extraction.
Prepare binding buffer calculating 10 mL / reaction .
| Reagent [Stock Concentration] | Final Concentration | Volume/reaction | |
| GuHCl (1 mol=95.53 g) | 5 M | 4.77 g | |
| UV HPLC-water | 6 mL | ||
| Isopropanol (100%) | 40% | 4 mL | |
| Total | 10 mL |
Weigh GuHCl and transfer into a glass bottle.
Safety information
If you want to clean the area where GuHCl was used, first use water and then bleach solution. Do not use bleach directly as it reacts with GuHCl to produce toxic chlorine gas!
Add UV-irradiated HPLC water (6 mL).
Note
This reaction is endothermic and the tube will become very cold. Be aware of the unusual 'slushy' way of dissolving.
Gently shake horizontally in order to get the salt dissolved. If necessary, apply short (00:00:10 ) bursts in microwave (~400W) keeping the tube slightly unscrewed. Wait until bottle cools down between microwave bursts.
Pipette isopropanol (4 mL) to reach the final reaction volume (10 mL ).
Prepare wash buffer by pipetting 40 mL ethanol to the wash buffer from the High Pure Viral Nucleic Acid kit following manufacturer's instuctions and make an aliquot calculating 900 µL / reaction .
Prepare TET elution buffer by making an aliquot of TE-buffer calculating 100 µL / reaction and pipette Tween-20 accordingly to reach 0.05 % (v/v) concentration to make 'TET'.
Note
Because Tween-20 is highly viscous, we dilute it 1:10 in UV-HPLC water, and use this 10% dilution to add Tween-20 to the TE-buffer
Irradiate all buffers with UV for 00:30:00 without the lids.
Note
UV irradiation can be done together with solutions from steps 1 (binding buffer), 4 (wash buffer), and 5 (TET buffer).
Store binding buffer in a fridge at 4 °C overnight for day 4.
Note
Label the bottle accordingly with the name, date and for the calculated amount of reactions. Buffer can be stored in a fridge for up to four weeks. Seal bottle with parafilm to avoid evaporation.
Dilute proteinase K powder (100 mg ) in 10 mL UV HPLC-water to a working concentration of 10 mg / 1 mL .
Note
Store proteinase K solution at -20 °C for use on day 2.
Day 1: Sample decontamination (DNA extraction room)
Day 1: Sample decontamination (DNA extraction room)
UV sterilize calculus. Irradiate samples for 00:01:00 , then shake the samples and irradiate for another 00:01:00 .
Note
Aim for 2-5 mg starting material.
Note
If the sample is in powder-form, irradiate in the original tube with the lid open. If the sample is in large pieces, place calculus in an aluminium foil boat in the crosslinker to maximize exposure.
If the calculus was in a boat for irradiation place it back in a 1.5 mL or 2 mL tube. Add 1 mL of 0.5 M EDTA to each sample, to remove surface contaminants.
Note
This step can be skipped if the purpose of the study is to detect infectuous pathogens, as these are more likely to be located on the outer layer of the calculus deposit (however, this may lead to a higher proportion of environmental contaminants in the data).
Vortex and incubate on rotator at room temperature for 00:15:00 .
Note
The incubation step can lead to some sample loss, as the calculus will start dissolving and releasing DNA. If the calculus is very powdery, the supernatant can be removed directly after vortexing, without any incubation. Otherwise the calculus solubilizes within the 15 min and you lose DNA while removing the EDTA.
Spin tubes 00:02:00 at 18500 x g .
Transfer supernatant to a separate tube, labelled "EDTA-wash". Store the "EDTA-wash" tube at -20 °C for potential future analyses.
Day 1: Decalcification (DNA Extraction Room)
Day 1: Decalcification (DNA Extraction Room)
Add 1 mL of fresh 0.5 M EDTA to each sample pellet.
Seal tubes with Parafilm, rotate Overnight (12-18h) with low overhead rotation speed (e.g., 12-16 rpm) at room temperature. If the tubes will be exposed to UV irridation, such as whole-room irridation for sterilization, be sure to cover them with foil or otherwise shield them.
Note
Post-incubation sample suspended in EDTA can safely be stored in a freezer (-20 °C ) before isolation and clean-up.
Day 2-3: Protein digestion (DNA Extraction Room)
Day 2-3: Protein digestion (DNA Extraction Room)
Remove Parafilm and make sure that the tubes have not leaked (if they have leaked, clean them).
Note
The appearance of the calculus will vary depending on its initial state. Larger pieces will have barely changed at all, while powder will be almost gone.
Briefly centrifuge tubes to remove any liquid from the lids.
Add 50 µL of 10 mg/ml proteinase K to each tube.
Vortex tubes, seal with Parafilm, and continue decalcification by rotating at low overhead rotation speed (e.g., 12-16 rpm) at room temperature for 24-48 hours.
Note
After starting the incubation, we recommend beginning preparations for day 4, such as pre-labelling the falcon tubes that will be used in step 17.
Note
Decalcification time can be adapted, so that the sample is completely decalcified before starting DNA isolation. After complete decalcification, the sample should appear buoyant and feathery, or disappear altogether.
Note
Post-incubation suspension can safely be stored in a freezer (-20 °C ) before isolation and clean-up.
Day 4: DNA isolation and clean-up (DNA Extraction Room)
Day 4: DNA isolation and clean-up (DNA Extraction Room)
Prepare cleaned workspace with all necessary reagents and equipment.
For each reaction prepare one 50 mL Falcon tube, one High Pure Extender Assembly (i.e. Falcon tube from kit containing funnel and purification column), two collection tubes from the kit, and one 1.5 mL LoBind tube for final elution step.
In every 50 mL Falcon tube pipette 10 mL binding buffer and 400 µL sodium acetate (UV-irradiated). Mix by inversion and measure pH (should be 5-6).
Note
Add more sodium acetate if the pH is too high. If the pH is too low you can add sodium hydroxide.
Remove parafilm from extraction tubes, then spin the tubes for 00:02:00 at 18500 x g to pellet calculus.
Note
If pellet is not solid, repeat centrifugation.
Pipette supernatant to respective 50 mL Falcon tube, mix contents by inversion. If pellet is too fragile, repeat centrifugation before transferring supernatant. Store the calculus pellet at -20 °C .
Pipette binding buffer/extract mix to High Pure Extender Assembly.
Spin at a maximum of 1500 rpm for 00:08:00
Safety information
This RPM is specific to a 50 mL Thermo Scientific TX-400 Swinging Bucket Rotor. As this is a swing rotor, the rpm value maybe inconsistent for other models. Therefore this value must be adjusted on a per-machine basis. Convert the rpm to rcf (g) and determine the appropriate rpm for your instrument.
Note
You can also turn the tube 180o after 00:04:00 to ensure the liquid does not get stuck on the inner rim of the funnel
Note
During this centrifugation step, we recommend preparing downstream steps, such as labelling of final elution tubes.
Pipette any liquids remaining in the funnel onto the column. Remove funnel from column and insert the column into a fresh 2 mL collection tube.
Note
The flow-through stays in the collection tube of the 50mL High Pure Extender Assembly and can be discarded.
Dry spin the column in the collection tube for 00:02:00 at 18500 x g .
Pipette 450 µL wash buffer from the High Pure Viral Nucleic Acid kit to the column, and spin at 8000 x g for 00:01:00 .
Remove column from the collection tube, discard the flow-through and the old collection tube, and put the column into a fresh 2 mL collection tube.
Repeat washing step once (go to step #24 ) reusing the collection tube, and discard flow-through.
Note
Discard flow-through in one of two following ways:
- Remove all liquid in the collection tube with a pipette, or
- Pour off the liquid into a fresh waste tube, and pat the rim of the collection tube dry on a paper tissue or towl. Use just one spot on the paper tissue per sample. Be careful not to touch the rim of the tube on the waste container. Be sure to clean the surface with DNA Away or bleach after discarding the paper.
Dry spin at 18500 x g for 00:01:00
Note
To ensure the liquid does not get stuck on the inner rim of the funnel, you can optionally spin for 00:00:30 , turn the tube 180o, and dry spin for another 00:00:30
Day 4: Elution (DNA Extraction Room)
Day 4: Elution (DNA Extraction Room)
Transfer column to a fresh 1.5 mL LoBind Tube, pipette 50 µL of Tris-EDTA-tween (TET) to the center of column, incubate for 00:03:00 on the benchtop, and spin 00:01:00 at 18500 x g to elute the DNA.
Note
Eluted DNA will be stored in this tube. Label tube on top and side accordingly.
Repeat elution step for a total elution volume of 100 µL TET.
Store the DNA extracts at -20 °C .