RPA Questions & Discussion

Molecular Diagnostics Protocols RPA
#1

Hi All,

There was some good interest in RPA in our last meeting so a thread for RPA might be what we need for in-between our meetings. Use this thread for all RPA questions, discussion, project updates :slight_smile:

#2

Notes on Homebrew Recombinase Polymerase Amplification Meeting - 2 June 2021

Attending - please add your name if I missed it

Agenda

  1. Introductions to selves and projects

  2. Why this meeting?

  3. Sharing notes about different challenges in different projects and places.

  4. Challenges with RPA and how to overcome them

  5. Peruvian team: expression of the proteins is good but the purification is showing some problems. UvsX-Histag and UvsY-Histag (C-terminal tagged) didn’t bind to the Ni resin. Not tested many conditions yet but very striking. Plasmids came from University of Cambridge, where they were purified and gave functional RPA. Aimorn also has a working set up for Ni purification and have functional RPA. Action: Smitha and Aimorn can share protocols on Reclone forum.

  6. Troubleshooting

  7. What was the buffer conditions? Will need to try some different conditions now we know other people have protocols.

  8. Peruvian team: would like to understand more about optimal reaction set up (specifically what are the components) to ensure everything is ordered.

  9. Troubleshooting

  10. pH of reaction: (Hongzhao) lower than 8 is not optimal. Around 8.3 is the optimum. Also +1 from Smitha. UvSY isoelectric point of 7.89 so avoid this or protein will precipitate. Anion exchange column seems to increase speed of reaction.

  11. Aimorn found problems with False Positive when using the buffer conditions from the purification, which is between 7 and 8 (buffer recommended by resin manufacturer).

  12. Protein purification yields are about 10-20 mg/ml (uvsY, uvsX) using both NiNTA resin and pre-packed fastflow columns. The lowest yield is obtained for gp32, which is critical for the assay.

  13. Smitha performed buffer exchange from purification to storage buffer using membrane dialysis. A key aspect is using glycerol (20-50%) in the buffer and a NaCl concentration of 250 mM to avoid precipitation.

  14. Protein purity is critical for reaction time (not rate of amplification). Adding an anion exchange step enables a faster reaction (40 min when only using Ni-NTA purification vs 30 min when adding the anion exchange purification step).

  15. How much protein to add to the RPA reaction? Smitha used this paper (key article!): Piepenburg O, Williams CH, Stemple DL, Armes NA (2006) DNA Detection Using Recombination Proteins. PLoS Biol 4(7): e204. https://doi.org/10.1371/journal.pbio.0040204

  16. Crowding agent: Aimorn used PEG 20000 as a crowding agent at 5%; Smitha used 20000 PEG and found that optimal matched the key paper above.

  17. Aimorn: LoD 100-1000cp of S gene

  18. UvsX and UvsY from China (never tested, molecular weights are different from that of the constructs we all have), Bsu and gp32 from NEB (works well). All enzyme are in mg/ml and hence easy to compare in-house to commercial.

  19. How to check activity?

  20. Do not have data on UvsX and UvsY to compare in protocol using FRET for Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51 (here and here) but plan to use this assay.

  21. Operating temperature can be anywhere from 37-40C for RPA, whichever works best for the primer sets.

  22. RPA is prone to contamination as much as LAMP, take all precautions.

  23. Outstanding questions

  24. Peruvian team asks: UvsX-Histag and UvsY-Histag didn’t bind to the Ni resin (in batch, not yet tested in column). Did you use the same constructs? What conditions favor Ni binding? Reply: the same constructs were used by Smitha and Aimorn. They do bind. Check the conditions.

  25. What are the components of the buffer for RPA reaction? How do you change from the purification buffer to the reaction buffer?

  26. Mix the concentrated protein in storage buffer into dilute reaction buffer, no buffer exchange.

  27. How do you change the purification to storage buffer

  28. Add glycerol (20-50%), pH8 Tris buffer and 250-500 mM NaCl to avoid precipitation.

  29. Used dialysis and protein concentration columns

  30. Next steps and actions

  31. Share protocols and then others can comment on things that you really have to pay attention to. Aimorn and Smitha to put working protocol on protocols.io and link in a thread on the Reclone forum

  32. Follow up meeting in a few months to reconvene? Deborah to invite everyone to a Reclone fortnightly meeting in 2-3 months - Note 18/08/2021 has been booked for this