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FAQs

Select the FAQs below to answer your questions or download them

1. Will PCR primers work in RPA?

2. Can I use my existing PCR probe?

3. How do I design RPA primers?

4. Can I multiplex TwistAmp® reactions?

5. How much of my primers do I have to use?

6. How do I reconstitute a lyophilised probe?

7. How much of my probe do I have to use?

8. Can I use TwistAmp® reactions for the quantification of template?

9. Can I add the components of the rehydration solution one at a time directly to the freeze dried reaction pellets?

10. Can I include the Magnesium-Acetate in my resuspension buffer?

11. Can I make a master-mix?

12. Can I perform a fluorescence end-point analysis of the amplification reaction?

13. Can I analyse the amplification product on an agarose gel?

14. How long do RPA primers have to be?

15. How far should RPA primers be apart?

16. How many primers do I have to screen?

17. Where can I get further details on primer design?

18. Do I need a probe for the primer screen?

19. Which conditions should I use for the primer screen?

20. Can I improve the performance of a given primer?

21. What makes a good primer?

22. What melting temperature should RPA primers have?

23. How do I select a probe?

24. Can I use intercalating dyes for real-time monitoring with the TwistAmp® technology?

25. Do my primers need any special purification protocols?

26. At what temperature should the TwistAmp® kit be used?

27. Do I need to clean up my RPA product before running it on a gel?

28. Do I need to dilute my RPA product before running it on a lateral flow strip?

29. I’ve tested some primers with a one type of TwistAmp® kit, will they work with another type of TwistAmp® kit?

30. Do I have to use a Twista® if I want to run a TwistAmp® reaction?

31. Can I just use labelled primers and a TwistAmp® Basic kit for lateral flow?

32. Does RPA work with biotinylated or fluorescently labelled oligonucleotides?

33. I sometimes see a sharp increase in fluorescence towards the end of my TwistAmp® exo reaction, is this normal?

34. Can I store the amplicons from my TwistAmp® reactions?

35. Can I use TwistAmp® Basic amplicons for TA cloning?

36. Can I add more/less Rehydration Buffer to reactions?


 

1. Will PCR primers work in RPA?

NO. Most PCR primers will not work in RPA because they are too short. Furthermore there is no established correlation between the qualities of an oligonucleotide as a RPA primer on the one hand and as a PCR primer on the other. See Appendix at http://www.twistdx.co.uk under Technical Resources for details about primer design.

 

2. Can I use my existing PCR probe?

NO. Most popular PCR probe systems are not suited for use with the TwistAmp® process. In particular those systems employing the 5’ to 3’ nuclease activity of polymerases cannot be used with the TwistAmp® system as such enzymatic activity is fundamentally incompatible with the RPA biochemistry.

 

3. How do I design RPA primers?

Good RPA primers are identified by a screening process described in detail in the Appendix at http://www.twistdx.co.uk.

 

4. Can I multiplex TwistAmp® reactions?

YES. It is possible to perform more than one amplification reaction simultaneously in the same tube. However, not all primer pair combinations will work equally well with each other in multiplexing and this format therefore requires careful primer design. Note that the total amount (nmols) of oligonucleotide in the reaction should not significantly exceed that stated in the protocol; if more than two amplification primers are used in a single reaction, then the maximum amount of primer has to be divided between all the oligonucleotides present. Monitoring multiple amplification events at the same time might also require different probes, and limitations on both, the detection equipment and the availability of compatible
fluorophores, have to be borne in mind.

 

5. How much of my primers do I have to use?

The recommended concentration of primers in the TwistAmp® Basic reaction is 480nM each. In the TwistAmp® exo, TwistAmp® fpg, and Twist Amp® nfo kits the recommended concentration of primer is 420nM each. However, the performance of some primer pairs can be improved by slightly varying their amounts in the reaction and a titration strategy (from 200nM to 600nM each) can be employed to identify the optimal concentration conditions for a given primer pair.

 

6. How do I reconstitute a lyophilised probe?

Please follow the oigonucleotide manufacturer’s instructions for the reconstitution and storage of the probes.

Typically the tube containing the lyophilised oligonucleotide will be spun briefly to collect the DNA at the bottom of the tube and an appropriate volume of T0.1E buffer (10mM Tris-HCI pH 8, 0.1mM EDTA) will be added to prepare a stock solution of 100μm. Allow the solution to stand for 10 minutes at room temperature and mix (vortex) for 10 seconds. Reconstituted oligonucleotide probes are typically stored at -20°C for the long term

 

7. How much of my probe do I have to use?

The recommended concentration of probe in the reaction is 120nM. However, some TwistAmp® reactions benefit from being used with slightly different amounts of probe. Testing different concentrations of probe (from 50nM to 150nM) will help to optimise the performance of probe based detection formats of a given assay.

 

8. Can I use TwistAmp® reactions for the quantification of template?

YES. The onset time of detectable amplification for a given assay will depend on the amount of starting template material – the more template copies there are to start with, the earlier the detection time will be. However, exploiting this ‘time-based’ quantification demands a careful experimental setup ensuring simultaneous initiation of compared reactions (e.g. through ‘Magnesium start’). The resolution of quantification is aided by a relatively ‘slow’ amplification reaction. Strategies to slow the rate of amplification (including the design of suitable primers) are discussed in the Appendix at www.twistdx.co.uk.

 

9. Can I add the components of the rehydration solution one at a time directly to the freeze dried reaction pellets?

NO. Adding one primer or probe before another will bias the formation of recombination filaments towards whichever oligonucleotide is added first. This is why primers and probes are added simultaneously.

 

10. Can I include the Magnesium-Acetate in my resuspension buffer?

YES. However the reaction components are activated as soon as Magnesium is added. By pipetting Magnesium Acetate into the reaction last, which we recommend, for example by adding it to the lids of strips and spinning it into the reactions, you ensure that reactions start simultaneously and you minimise the risk of cross contamination or RPA products being produced in any reaction residues left in your tips.

 

11. Can I make a master-mix?

YES. If you wish to set up multiple reactions, you can make a master mix. If you are screening different DNAs, the resuspension buffer, primers and probe if used can all be mixed together and added to freeze dried reactions to resuspend them. Different DNAs can then be added to reactions before they are started with MgAc as usual. If you are performing a primer screen, it is possible to make a master mix with the resuspension buffer, template DNA, probe (if used) and one of the primers. This should be aliquotted into 1.5 ml tubes and the variable primers added. Only once both primers are present should the freeze dried reactions be resuspended or the formation of recombination filaments will be biased towards the first oligonucleotide added.

 

12. Can I perform a fluorescence end-point analysis of the amplification reaction?

YES. The measurement of change of fluorescence can also be used as an endpoint read-out. In this case the fluorescence at the start and at the end of the reaction is compared: an increase of fluorescence signifies a successful amplification event.

 

13. Can I analyse the amplification product on an agarose gel?

YES, UNLESS you are using the TwistAmp® exo kit. The exonuclease present in the reaction mixture will digest most of the amplification product once amplification has ceased. For the analysis of amplicon, use the TwistAmp® Basic kit. It is also possible to analyse products on gels generated from the TwistAmp®nfo and TwistAmp® fpg kits. NB The TwistAmp®nfo kit can be used with TwistAmp® exo probes if you would like to run these on a gel, although the kinetics will be slower.

 

14. How long do RPA primers have to be?

RPA primers should be at least 30 nucleotides long. Typically, primers are between 32 and 35 nucleotides long. In some cases primer can be shorter than 30 residues, but this usually slows overall amplification kinetics. See http://www.twistdx.co.uk for details about primer design.

 

15. How far should RPA primers be apart?

This depends on the application and need. In general we recommend that under the conditions of the standard TwistAmp® kits the amplicon generated by two RPA primers should typically be no longer than about 500bp. There is probably no lower limit to the size of RPA products, but the minimum size of RPA primers requires that amplicon will typically be longer than about 80bp. For the most rapid real-time kinetics the final amplicons should ideally be 100-200 base pairs. Under specialised conditions amplification products of as much as 2 kilobases have been generated. However, the standard TwistAmp® kits do not readily permit the generation of such large amplicons. See Appendix at http://www.twistdx.co.uk for details about primer design and approaches to achieve larger amplicon sizes.

 

16. How many primers do I have to screen?

This depends on the sensitivity specifications of the assay. For example, if you only need to detect 1000 molecules per reaction or more, then most primer pairs will be adequate. For very high sensitivity assays, the best way to find good RPA primers is by performing a systematic screen. Typically the number of tested primer pairs will lie between 10 and 20 initially. The more oligonucleotides are tested, the greater the chance of finding good primer pairs capable of detecting single molecules with rapid kinetics.

 

17. Where can I get further details on primer design?

For further details on primer design, see the Appendix at www.twistdx.co.uk under Technical Resources / Instruction manuals / Appendix.

 

18. Do I need a probe for the primer screen?

NO. Any detection method can be used to evaluate and compare the performance of potential primer pairs. However, real-time monitoring of the amplification with a detection probe has proven to be the fastest and least laborious method for screening high sensitivity primers.

 

19. Which conditions should I use for the primer screen?

Ideally the conditions of the screen should mimic as closely as possible the conditions expected in the final assay (approximate template copy number, sample purity, depth of multiplexing, etc.).

 

20. Can I improve the performance of a given primer?

Small changes in the sequence of a primer can improve RPA performance. Any given primer can be optimised by slightly varying its length (by single nucleotides) and position (keeping the length but shifting their location in 1bp increments), and re-testing its activity.

 

21. What makes a good primer?

The precise rules for this are not yet known, this is why primer screening is so important.

 

22. What melting temperature should RPA primers have?

As RPA reactions are performed at a constant temperature and under conditions under which the melting behaviour of DNA is drastically altered by DNA melting proteins, conventionally calculated melting points are not directly applicable to the system.

 

23. How do I select a probe?

If using one of the TwistAmp® kits suitable for probe use, follow the guidelines described in the Appendix at http://www.twistdx.co.uk for the design of detection probes. Note that there are some sequence restrictions in choosing the position of probes within a target using the preferred TwistAmp® exo Probe system. If this presents unreasonable limitations TwistDx can help you design alternative detection strategies. The alternative TwistAmp® fpg Probe system is far more amenable to flexible design, but does not always yield as strong fluorescence signal as TwistAmp® exo Probe.

 

24. Can I use intercalating dyes for real-time monitoring with the TwistAmp® technology?

YES. It is possible to use intercalating dyes to quantify and monitor the TwistAmp® reactions in progress. However, as with PCR, the dye typically binds to any double-stranded DNA, so primer noise can generate false positive signals. Moreover, the exonuclease present in the TwistAmp® exo kit will digest most of the amplification product during the reaction, and the use of intercalating dyes is not recommended with these kits.

 

25. Do my primers need any special purification protocols?

When performing a primer screen, one does not normally need to use specially purified oligonucleotides. However, as with use in other techniques, we have noted batch-to-batch variations in the quality of primer preparations. For applications where consistency is critical, we would therefore recommend the use of more purified primers once good primers are identified.

 

26. At what temperature should the TwistAmp® kit be used?

The standard TwistAmp® kits are configured to operate in the temperature range of 37°C - 42°C. At higher temperatures the system will be compromised as the enzymes progressively lose full activity. The RPA process itself can, under appropriate conditions, be performed at much lower temperatures, but the supplied formulations of the TwistAmp® kits are optimised for high kinetic rate and not generally compatible with protocols using temperatures below the recommended range.

 

27. Do I need to clean up my RPA product before running it on a gel?

Yes. The crowding agent and proteins in an RPA reaction interfere with normal agarose gel electrophoresis, so you will probably get a smear, rather than a clean band if you do not clean up your reaction.

 

28. Do I need to dilute my RPA product before running it on a lateral flow strip?

Yes. The crowding agent and proteins in RPA reactions can interfere with the antibodies on a lateral flow strip, so you can get nonspecific binding and false positive signals if you do not dilute them sufficiently.

 

29. I’ve tested some primers with a one type of TwistAmp® kit, will they work with another type of TwistAmp® kit?

Maybe. If you have developed primers using a TwistAmp® Basic kit, you will also need to include a probe if you are going to use a TwistAmp® exo or nfo kit. This and other factors mean that
an optimal primer combination for a gel-based approach vs a fluorescence or lateral flow approach may not be the same. In general we have found that switching from TwistAmp® exo fluorescence monitoring to TwistAmp® nfo for lateral flow monitoring using a similar sequence probe gives fairly reliable results. The best primer/ probe combination for fluorescence monitoring is however not necessarily the primer combination that will give the most attractive gel-based result and vice versa.

 

30. Do I have to use a Twista® if I want to run a TwistAmp® reaction?

No. For TwistAmp® Basic and nfo kits, any device that can hold a steady temperature of 37-42°C is fine. For real-time reactions using TwistAmp® exo kits, any plate reader or real-time thermal cycler that can excite and detect the fluorophores you are using and hold a steady temperature of 37-42°C is fine. NB heated lids should be switched off when running RPA reactions.

 

31. Can I just use labelled primers and a TwistAmp® Basic kit for lateral flow?

Yes. You can use two modified primers for lateral flow if you wish, but we recommend using a probe and a TwistAmp® nfo kit. This is because, as with PCR, RPA is subject to primer-dimer formation, so you can get primers cross reacting and giving false positives if you do not have perfectly designed primers. TwistAmp® nfo probes avoid this problem because they are blocked and cannot be extended to create probe-primer dimers. The nfo enzyme recognises and cuts the abasic site (THF) in the probe only when it has bound to it’s complementary strand. Cutting the abasic site means that the blocked end of the probe can fall off and the probe can act as a primer, thereby generating a product that can be captured by a lateral flow strip. So, only if you have your desired amplicon can the probe bind, be cut, and extend to form a product with the opposing biotinylated primer.

 

32. Does RPA work with biotinylated or fluorescently labelled oligonucleotides?

Yes. Biotinylated primers or ones with fluorophore should work the same as unmodified primers in an RPA reaction. We have yet to see any differences.

 

33. I sometimes see a sharp increase in fluorescence towards the end of my TwistAmp® exo reaction, is this normal?

Yes. The polymerase and exonuclease III in TwistAmp® exo reactions are in competition. As the reaction runs out of energy, the exonuclease III starts to dominate. This leads to faster probe cleavage and a jump in fluorescence signal.

 

34. Can I store the amplicons from my TwistAmp® reactions?

Yes, if you have used TwistAmp® Basic or nfo reactions, you can store your amplicons at 4°C for short periods of time and -20°C for longer periods of time.

No, if you have used TwistAmp® exo reactions. The exonuclease III is likely to digest the amplicon if the reaction is not processed rapidly to inactivate any such activity.

 

35. Can I use TwistAmp® Basic amplicons for TA cloning?

The polymerase used in TwistAmp® Basic reactions does not have an editing function, so this should be possible. However, we have not tested this ourselves.

 

36. Can I add more/less Rehydration Buffer to reactions?

NO. Adding more or less Rehydration Buffer than is recommended can have a detrimental impact on how an RPA reaction works. The buffer contains ingredients that are necessary for RPA reactions, so adding more or less buffer will change their final concentration in a resuspended pellet.

 


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