Feb 21, 2025

Public workspaceUSER II treatment of ancient DNA extracts

DOI
https://dx.doi.org/10.17504/protocols.io.eq2ly6qrwgx9/v1
  • Alba Rey-Iglesia1,
  • Deon de Jager1,
  • Vanssy Li1,
  • Andrea A. Cabrera1,
  • Michael V. Westbury1,
  • Eline D. Lorenzen1
  • 1Globe Institute, University of Copenhagen
  • Lorenzen Lab
Deon de Jager
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2ly6qrwgx9/v1
Protocol Citation: Alba Rey-Iglesia, Deon de Jager, Vanssy Li, Andrea A. Cabrera, Michael V. Westbury, Eline D. Lorenzen 2025. USER II treatment of ancient DNA extracts. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly6qrwgx9/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: February 07, 2025
Last Modified: February 21, 2025
Protocol Integer ID: 119695
Keywords: Ancient DNA, DNA extraction, USER treatment, UDG treatment, USER-II treatment, Uracil, Paleogenetics, antarctic thermolabile uracil dna glycosylase, thermolabile user ii enzyme, enzymatic removal of uracil base, lyase activity of endonuclease iii, enzymatic removal, user ii treatment of ancient dna, user enzyme, enzyme, uracil base, phosphodiester backbone, endonuclease iii, ancient dna fragment, ancient dna, dna
Abstract
Enzymatic removal of uracil bases from ancient DNA fragments. "Antarctic Thermolabile Uracil DNA glycosylase (UDG) catalyzes the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. The lyase activity of Endonuclease III breaks the phosphodiester backbone at the 3´and 5´ sides of the abasic site. In addition to generating a different a 3´-terminus than USER Enzyme (a 3´-phospho-α, β-unsaturated aldehyde versus the 3´phosphate left by USER Enzyme, NEB #M5505), Thermolabile USER II Enzyme (NEB #M5508) can also be completely heat inactivated after 10 minutes at 65°C." - https://www.neb.com/en/products/m5508-thermolabile-user-ii-enzyme?srsltid=AfmBOooXGOM1bvBPvVpj3P3CCKKOiRKUrQ-PfReg2m_cax4zrM3j8pN4.

Protocol materials
ReagentThermolabile USER II Enzyme - 250 unitsNew England BiolabsCatalog #M5508L
ReagentBuffer PEQiagenCatalog #19065
ReagentBuffer EBQiagenCatalog #19086
Reagent1.5 mL LoBind tubes EppendorfCatalog #022431021
ReagentMonarch DNA Cleanup Columns (5ug) - 100 columnsNew England BiolabsCatalog #T1034L
Troubleshooting
Before start
  • UV-treat buffers (modified Buffer PB, Buffer PE, and Buffer EB/EBT) before starting for 10 min in a UV box.
  • Do not UV Eppendorf/PCR tubes.
  • PE buffer: Add Molecular Grade Ethanol as indicated on the bottle before use.
  • Clean all surfaces with 5% bleach solution followed by 70% ethanol before and after use.
  • Clean all equipment with 70% ethanol before and after use.
Buffer preparation
See following protocol for buffer preparation protocols.
Protocol
NAME

Ancient DNA Extraction from Bones and Teeth

CREATED BY
Vanssy Li

Required buffers for this protocol:
  • Buffer PE
  • Modified Buffer PB
  • Buffer EB - if using LoBind tubes
  • Buffer EBT - if not using LoBind tubes (Buffer EB + Tween-20)
Incubation with USER II
In a PCR tube, add 4.8 µL ReagentThermolabile USER II Enzyme - 250 unitsNew England BiolabsCatalog #M5508L to 27.2 µL DNA extract, for a final reaction volume of 32 µL.

Optional: Or add 2.4 µL USER II enzyme to 13.6 µL DNA extract, for a final reaction volume of 16 µL, if you want to keep some of the DNA extract as untreated.
Optional
In a thermocycler, incubate the reaction for 3 hours at 37°C then cool down to 10ºC. Can be left overnight at 10ºC.
Incubation
Clean-up
Clean up using ReagentMonarch DNA Cleanup Columns (5ug) - 100 columnsNew England BiolabsCatalog #T1034L


Note
  • Do not touch the film of the spin column when adding liquids.
  • Pay attention when adding small volume of liquid. Make sure the liquid do not attach on the wall.
  • As of 31 December 2024 the "Monarch DNA Cleanup Columns (5ug) New England Biolabs Catalog #T1024L" have been discontinued and replaced by "Monarch Spin Columns S1A and Tubes New England Biolabs Catalog #T2037L".


Add 450 µl modified Buffer PB to the spin column.
Add the entire USER II-DNA reaction (32 or 16 µL) and mix by pipetting or inversion.
Centrifuge at 8,000 RPM in a benchtop centrifuge for 1 min, discard flow through.
Centrifigation
Add 650 µl ReagentBuffer PEQiagenCatalog #19065 to the spin column.

Centrifuge at 8,000 RPM for 1 min, discard flow through.
Centrifigation
Centrifuge at max speed for 1 min to dry the column.
Centrifigation
Place the spin column in a fresh 1.5 mL Eppendorf tube. Use Reagent1.5 mL LoBind tubes EppendorfCatalog #022431021 , if available, but if not available, then use EBT buffer instead of EB buffer for the final elution (Step 4.8).

Add 20 µL ReagentBuffer EBQiagenCatalog #19086 to the spin column. If not using LoBind tubes, add 20 uL EBT buffer instead.

Incubate for 5 mins at room temperature to allow the DNA to elute from the column and dissolve in the buffer.
Incubation
Centrifuge at max speed for 1 min.
Repeat Steps 4.8-4.10.
Discard the spin column and store the eluted DNA at -20ºC.
Protocol references
Hofreiter, M., Jaenicke, V., Serre, D., Haeseler, A. v., & Pääbo, S. (2001). DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA. Nucleic Acids Research, 29(23), 4793-4799. https://doi.org/10.1093/nar/29.23.4793