Frequently asked questions
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. For RT kits, we recommend that users run reactions at 40°C as the reverse transcriptase performs better at slightly higher temperatures.
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.
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.
Yes, unless you are using the TwistAmp® exo kit or exo RT 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.
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.
Yes, if you are using TwistAmp® Basic, nfo or fpg reactions. 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.
No, if you are using TwistAmp® exo or TwistAmp® exo RT reactions. In the presence of Mg, the exonuclease can attack the primers and probes before they are protected by the recombinase and singlestranded binding proteins. This may cause a high fluorescence base line in your reactions.
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 its 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 labelled primer.
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.
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.
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.
The Twista® is not Windows 8 compatible, so you will have to install an emulator of an earlier version of Windows (XP, Vista or 7) in order to get it to run.
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 (RT) reactions. The exonuclease III is likely to digest the amplicon if the reaction is not processed rapidly to inactivate any such activity.
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. This noise is made worse as the dye is monitored at a low temperature – in PCR assays the temperature is often such that primer dimers are melted when intercalated dyes are measured. 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.
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.
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.
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 assay design manual .
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.
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.
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.
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.
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.
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.
Reproducibility can obviously be a problem when pipetting small volumes of liquid sub-components as variable component ratios could arise. It is advisable to make a master mix to include the Core Reaction Mix, E-mix etc. which should make things more reproducible. If it’s not feasible to make a master mix, then reverse pipetting can also improve reproducibility.
Please follow the oligonucleotide 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.
If using one of the TwistAmp® kits suitable for probe use, follow the guidelines described in the assay design manual 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.
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 assay design manual for details about primer design and approaches to achieve larger amplicon sizes.
We recommend two possible variant approaches to making master mixes: One for when trying different oligo combinations, and one for testing different templates once the oligonucleotides are chosen. Changing the order of addition for these reactions can lead to sub-optimal RPA reactions. If using the TwistAmp® Liquid exo kit, it is best to keep the 50x Exo reagent separate from oligos/template until they have been coated in ssDNA binding proteins (present in the Core Reaction Mix), or they may be digested. MgAc should always be added last as it starts the reactions instantly and will begin the consumption of ATP even if no template has been added. Oligonucleotides should all be introduced to the Core Reaction Mix simultaneously to prevent preferential binding of the oligonucleotide introduced first.
To take full advantage of the speed and sensitivity of RPA, we recommend that customers use primers that are at least 30 nucleotides long. Typically, primers are between 32 and 35 nucleotides long. Shorter, PCR-length, primers can be used, but these may lack the speed and sensitivity of longer primers.
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.
Reproducibility of replicate reactions can be optimised by considering the following: For low copy numbers of template, variable amplification could be because the limit of detection for an assay is being reached or because of differential agitation of reactions (with poorly agitated reactions not amplifying as well). Alternatively, the recombinase and ancillary protein in the 20x Core Reaction Mix are known to co-precipitate in 50% glycerol when stored at low temperatures. Warming the 20x Core Reaction Mix to room temperature and mixing will return them into solution, with no loss of function and could improve reproducibility. Variable amplification can also result from insufficiently mixed master mix. Users are advised to check post agitation that the 20x Core Reaction Mix is homogenous before adding it to reactions.
We recommend using 1.8mM (total) dNTPs as a starting point, however, users may choose to optimise the concentration as part of their specific assay development – sometimes other amounts (e.g. 1-12mM) may be preferred.
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.
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.
If positive results are seen in no template controls it is commonly caused by contamination, particularly, if the positive control oligonucleotides provided in the kit display false positives when no template is added. Contamination can occur if you are pipetting high copies (10^6 /µl or above) of template in the same area as carrying out RPA testing. We strongly recommend having separate pre and post amplification areas i.e. where you set up reactions, and where you open them for analysis. Other contamination control measures that we recommend include aliquoting primers and probes with different pipettes, and in a different lab, to where you perform RPA experiments. When conducting initial RPA assay testing, we suggest working with low amounts of template (at ~50 copies/µl) and advise against opening anything with more than 10,000 copies/µl anywhere near where you do set-ups if possible. Another good contamination control measure we would suggest adopting is to add the MgOAc to the lids, and spinning it in to start your reactions. It is essential to apply strict contamination control measures when using any end point detection methods where tubes of reactions are opened post amplification. To rule out, or clear contamination we suggest decontaminating work areas with 10% bleach, and using fresh reagent aliquots (buffer, MgOAc, etc). You may need to repeat this bleach clean-up a few times before removing contamination completely.
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.
Oligos, 2x Reaction Buffer, water, dNTPs and 10x E-mix can be combined and stored on ice for a minimum of hours, and over-night in many cases (any effects on performance will be assay dependent and should be assessed independently by users). Once the 20x Core Reaction Mix is added short term storage on ice (assay dependent again) should also be possible.
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. Switching from a DNA-detecting assay, to an RNA-detecting RT assay, has the added complication that the reverse-transcriptase may favour different primers to the polymerase. As such we recommend developing RNA assays using an RT system on RNA template and not trying to switch to an RT kit after optimising with a DNA kit.
Yes, kits are fine to be at ambient temperatures for 2 – 3 weeks and should still work well. If you are unsure, please run a control test and send us the results, and we will be able to confirm that this is the case. For long term storage we suggest storage at -20˚C.
The recombinase and ancillary protein present in the 20x Core Reaction Mix are known to sometimes reversibly co-precipitate in 50% glycerol when stored at low temperatures. Warming the 20x Core Reaction Mix to room temperature and mixing will return them into solution, with no loss of function.
Some small amount of the phosphocreatine in E-mix can occasionally form a precipitate. This should not affect RPA reactions as there is a large excess of phosphocreatine in the E-mix. Vortex the tube to resuspend precipitate and spin down any remaining precipitate before using the supernatant.
Picolitre volume RPA reactions have been shown to work in published literature http://dx.doi.org/10.1371/journal.pone.0153359 .Obviously, this is subject to having the mechanism to generate such a volume.
The precise rules for this are not yet known, this is why primer screening is so important.
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.
Delivery should be within 3 weeks of placing an order or payment. The Buyer will receive a notification of an expected delivery date upon placement of order. Upon shipping the product to the buyer, the Buyer will receive a notification from the courier company with your tracking number. The Company uses FedEx for international shipping.
This is normal for TwistAmp® Liquid reactions, and is not a problem. RPA reactions form emulsions which can sometimes be observed by the naked eye.
If you are combining your RNA template with MgOAc before adding it to your reactions, you may be stabilising RNA tertiary structures that inhibit the reverse transcriptase. Adding the RNA to your reaction mixture either before you add the MgOAc, or spatially separating it (i.e. adding RNA and MgOAc to opposite sides of your reaction vessel or lid) and adding it simultaneously may improve performance.
For further details on primer design, please see the assay design manual
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.).
In practise, it makes no difference which way round the labels are used i.e. which label is on the probe, and which one is on the primer. However, for ease of further test development, it may prove useful to put the hapten for the conjugate antibody on the probe (FAM for Milenia, Biotin for PCRD). This way, users who proceed to use a two line strip for multiplexing, can change which line on the strip they detect at by ordering a new labelled primer rather than ordering a new probe.
Yes. We have recently confirmed that primers of lengths typical for use in PCR (e.g. 18-23 nucleotides) work just fine in RPA in many cases. Note though that kinetics can be a little slower, so if you wish to take full advantage of the potential of RPA for rapid amplification within 10-15 minutes we recommend using slightly longer primers (as well as shorter amplicons). Also, note that at this time there is no formally established correlation between the elements which dictate performance of an oligonucleotide as a RPA primer on the one hand and as a PCR primer on the other.