Jan 15, 2025
Version 1
Preparation of single-stranded DNA capture probes V.1
- 1Max Planck Institute for Evolutionary Anthropology
- MPI EVA Ancient DNA Core Unit
Document Citation: Sarah Nagel, Birgit Nickel, Matthias Meyer 2025. Preparation of single-stranded DNA capture probes . protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7mr89gwz/v1
License: This is an open access document 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
Created: October 30, 2024
Last Modified: January 15, 2025
Document Integer ID: 111255
Funders Acknowledgements:
Max Planck Society
Grant ID: -
Abstract
This document describes the preparation of biotinylated single-stranded DNA capture probes for use in hybridization capture as described in Fu et al. 2013. It starts from amplified probe libraries carrying the following adapter sequences: ATAGGGATCGCACCAGCGTGT-variable-probe-sequence-CACTGCGGCTCCTCATCCACG. These probe libraries can be generated using custom-designed capture arrays such as one million feature arrays from Agilent Technologies (see supporting information in Fu et al. 2013; section 3.2.3.) or custom-synthesized oligonucleotide pools, for example from GenScript ('GenTitanTM Oligo Pools') or Integrated DNA Technologies ('oPools').
References
Fu, Q., Meyer, M., Gao, X., Stenzel, U., Burbano, H. A., Kelso, J., & Pääbo, S. (2013). DNA analysis of an early modern human from Tianyuan Cave, China. Proceedings of the National Academy of Sciences of the United States of America, 110, 2223-2227.
Note
This protocol describes the preparation of at least 80 µg of single-stranded probe using 96 parallel single primer extension reactions. The required template for probe production is a purified PCR products of a probe library adjusted to a concentration of 100 ng/µl. In the Ancient DNA Core Unit of the MPI-EVA, probe library templates are labeled with a probe name (e.g., AA213) followed by 'short amp'.
Materials
| Reagent/consumable | Supplier | Catalogue number | |
| Reagents | |||
| Herculase II Fusion DNA Polymerase Kit | Agilent | 600679 | |
| Oligonucleotide APL2 * | Sigma/Merck | - | |
| 25 mM each dNTP | Thermo Fisher Scientific | R1122 | |
| Water, Milli-Q | Merck | - | |
| SPRI beads (38% PEG) | self | ||
| Ethanol 70% (v/v) | self | ||
| EB buffer | self | - | |
| AAxx short amp library (100 ng/ µl) | self | - | |
| Consumables | |||
| 50 ml Falcon tube, without stand | Greiner Bio-one | 227261 | |
| 50 ml glass pipette | Greiner Bio-one | 768180 | |
| Eppendorf twin.tec PCR plate 96, semi-skirted | neoLab Migge | VB-0398 | |
| 15 ml Falcon tube, without stand | Greiner Bio-one | 188261 | |
| PCR cap strips | Eppendorf | 0030124839 | |
| Eppendorf safe-lock tubes, 1.5 ml | neoLab Migge | VB-0352 | |
| Eppendorf safe-lock tubes, 2 ml | neoLab Migge | VB-0353 | |
* Order oligonucleotide APL2 at 0.2 µmol synthesis scale (Sigma/Merck, HPLC purified). Dissolve in water at a concentration of 100 µM. Prepare a 10 µM working dilution in water. Sequence: 5'-biotin-CGTGGATGAGGAGCCGCAGTG-3'
† See document in the Appendix for the preparation of SPRI beads.
‡ See document in the Appendix for the preparation of EB buffer.
Equipment
- Thermal cycler for PCR strip tubes (e.g. Veriti 96-Well fast Thermal Cycler, cat. no. 4375305)
- Rotator (e.g. Phoenix Instrument, RS-TR05, cat. no. XK30.1)
- 50 ml magnetic separation rack for Falcon tubes (e.g. NEB, cat.no. S1507S)
- 1.5 ml magnetic separation rack (e.g. Bio-Rad, cat.no. 1614916)
- Serological pipette controller (e.g. battery-powered pipetting aid ROTILABO, cat. no. TC16.1)
Protocol
1. Prepare a master mix by combining the reagents listed in the table below in a 15 ml Falcon tube. Do not add the enzyme yet. Mix properly by vortexing and spin down. Add the polymerase, mix briefly by flipping the tube with a finger, and add 100 µl of the mix to each well of a 96-well PCR plate.
| Reagent | Volume (µl) per reaction | Volume (µl) in master mix (100 rxn) | Final concentration in reaction | |
| Water | 65.50 | 6,550 | ||
| 5x Herculase II reaction buffer | 20 | 2,000 | 1x | |
| 10 µM APL2 | 10 | 1,000 | 1 µM | |
| 25 mM each dNTP | 1 | 100 | 0.25 mM | |
| Herculase II fusion polymerase | 1.50 | 150 | N/A | |
| Template: AAxx short amp 100 ng/µl | 2 | 200 | 2 ng/µl | |
| sum | 100 | 10,000 |
2. Seal the plate with PCR cap strips and centrifuge briefly. Place the plate in a thermal cycler and incubate under the following conditions:
| Step | Temperature (°C) | Time | |
| Initial denaturation | 95 | 2 min | |
| Per cycle denaturation | 95 | 30 s | |
| per cycle annealing | 60 | 30 s | |
| per cycle extension | 72 | 30 s | |
| Go to step 2, 19 times | |||
| Final extension | 72 | 5 min | |
| Hold | 8 | forever |
3. Transfer 20 ml of SPRI bead suspension (38% PEG) to a 50 ml Falcon tube. Add the PCR products from the plate to the Falcon tube and mix on a rotator for 60 min at room temperature.
4. Centrifuge the Falcon tube briefly and place it on a magnetic rack. Let it stand for about 1.5 hours.
5. Remove and discard the supernatant using a serological pipette. To wash the beads, leave the tube on the magnetic rack and add 50 ml of 70% Ethanol without disturbing the bead pellet. Let stand for approx. 2 min and remove the Ethanol with a pipette. Repeat this step for a total number of two wash steps.
6. Remove any residual Ethanol with a pipette and allow the beads to air dry for 1 - 1.5 hours.
Note
[Note]
Make sure the bead pellet is not completely drying out (no formation of cracks should be visible).
7. Remove the Falcon tube from the magnetic rack and place it horizontally on the bench. Add 300 µl EB buffer directly on top of the bead pellets. Do not move the Falcon tube. Wait for 5 min, lift the tube, close it and briefly centrifuge.
8. Resuspend the bead pellet in the bottom of the tube using a pipette. Transfer the bead suspension to a fresh 2.0 ml Eppendorf tube (tube #1).
9. Add another 50 µl EB buffer to the wall of the Falcon tube to rinse residual beads from the tube wall. Repeat this step.
10. If necessary, briefly centrifuge the Falcon tube to collect the liquid at the bottom. Resuspend beads with a pipette and transfer the suspension to the 2.0 ml Eppendorf tube (tube #1).
11. Briefly vortex tube #1 and let it stand for 5 min. Briefly centrifuge tube #1.
12. Place tube #1 into a magnetic rack and let it sit for 5 min.
13. Transfer the supernatant to a fresh 2.0 ml tube (tube #2).
14. Add 50 µl EB buffer to the the bead pellet in tube #1 and resuspend the pellet by vortexing. Let tube #1 stand for 2 min and centrifuge it briefly.
14. Place tube #1 into a magnetic rack and let it sit for 5 min.
15. Transfer the supernatant to tube #2 already containing the eluate from step 13. Briefly centrifuge tube #2 if necessary.
16. Place tube #2 in a magnetic rack and let it sit for 5 min to pellet any residual beads.
17. Transfer the final eluate to a fresh 1.5 ml tube (tube #3).
18. Measure the concentration of the final probe solution using the NanoDrop device at OD 33.
19. Dilute the probes in EB buffer to the concentration specified in the hybridization capture protocol. Required concentrations may vary depending on the characteristics of the probe set (e.g., mitochondrial vs. nuclear targets).
Note
[Note]
Since the final probe solution might be slightly foamy, it is recommended to add 2 µl of the eluate to the measuring pedestal of the NanoDrop device.
[Note]
The concentration of your final probe solution is expected to be greater than 200 ng/µl.
Note
[Labeling]
Label the final probe tubes with ssAAxx probe name, APL2, the concentration, the date and your initials.
12. Store the probe at -20°C until use.
Appendix
Document
NAME
SPRI beads, variable PEG concentration
CREATED BY
Ancient DNA Core Unit
Document
NAME
EB buffer
CREATED BY
Ancient DNA Core Unit