hello everybody,

In my humble opinion, DDPCR is another device that takes the assay planning and assay establishment to the manufacturer. You just take the suitable kit from the supplier, mix it with your material, put it in the device and the software does everything for you. Don´t get me wrong, its fine that scientists have less and less "stupid work" to do. High throughput (ht) techniques are great but very expensive. Not many labs can afford to go on ht, and even, it doesn't make more sense for every application to go on ht. More and more papers and fund proposals have to include ht techniques in order to be accepted, and as a result DDPCR will be the key for future science.

On the other hand, establishing new qPCR assays in the lab is so easy and inexpensive, with the result that it will be the way to go for specific questions, pretrails, and pilot tests.

qPCR cycler are not just used for dna/rna/cdna amplification, but also for methylation analysis and HRM. So, a qPCR device is versatile and covers a wide range of applications.

Conclusion: DDPCR will never replace qPCR. Labs will use both or just the qPCR. Labs without a DDPCR (and of course other ht devices) will outsource this to service supplier.

Why should it. Both can do the same things and both and do different things. Just a matter of what you need to do.

Thanks Joshua for your concise reply. As you know technologies are going cheaper hence the financial aspect will be the decisive factor as to which technology you chose to do what; hence my question. As far as I know the relative advantage of DDPCR over qRT-PCR is that it requires no knowledge of the initial target concentration and any target is quantifiable regardless of the quantity, hence it has q lower detection limit let alone the cheaper cost involved.

Mourad, any well-optimized PCR is able to amplifiy just a single molecule up to detectable amounts. DDPCR is limited only by the Poisson error law, whereas RTPCR additionally has to cope with amplification efficiency, background drifts, and measurement errors. That DDPCR circumvents the requirement of constant and identical amplification efficiencies seems to me the most important aspect.

The costs will mainly decide short-term. Long-term I think that DDPCR will rule because of the more direct way of quantification.

DDPCR may rule long term but that long term is a looooooonngggg way away. It will take many years for current machines become obsolete and replaced. Also, the purchaser of the DDPCR machine must agree that it is better, that will be the toughest sell. Change is difficult for most people. I do agree that once costs come down and it is reported more in literature it will win but it will take a long time. A few years ago I still worked with a thermocycler that was 10 years old and still going strong.

I think that digital Recombinase Polymerase Amplification (DRPA) will replace DDPCR - why wait around when you can do your reaction in 5-10 minutes? http://www.ncbi.nlm.nih.gov/pubmed/21476587

This special procedure is hard to optimize and (as i understuud) requires circular templates. However, using any kind of isothermal amplification technique will in fact be a step foreward. The biochemistries should work in DDPCR instuments as well.

No need for circular templates for RPA - it uses two primers and a probe like PCR. Enzymes replace thermal cycling so you run your reaction at a steady temperature of 37-40C: http://www.twistdx.co.uk/our_technology/

The only difference is the primers are a bit longer (30-35 bases) and because RPA uses a strand-displacing polymerase and single-strand DNA binding proteins you have to use a TwistAmp probe rather than a TaqMan probe or molecular beacon. TwistAmp exo probes are just oligos with a fluorophore and a quencher separated by an abasic site that is cleaved when the probe binds to your target.

I don't think it's very difficult to optimise an RPA reaction, you just buy some freeze dried reactions, add some primers and your template and find a good combo. I think you're getting confused with LAMP, which is tricky.

There are some RPA publications here if you want to check them out: http://www.twistdx.co.uk/resources/publications/

So Mathew: in RPA there will be no need for a thermal cycler as rxns will take place enzymatically at 37°C?; any regular incubator will do just in restriction enzymatic reactions?. How expensive a one reaction will it be?.

That's correct Mourad, although I prefer to run my reactions at ~40C as they're a little bit faster and with our reverse transcriptase system we recommend 40C as well (the RT likes things a bit warmer). Obviously if you want real-time data, you will need some sort of fluorescent detection system, but it can be a plate reader or any real-time PCR machine - it's platform agnostic. If you don't need real-time data, you could just hold the tube and heat it that way, but something with temperature control is more reproducible. I'm afraid that reactions are a bit more expensive than PCR as production is still being ramped up and it's a bit of a niche product at the moment, but everything (dNTPs, enzymes etc) is freeze dried in, you just need to provide your oligos and your template. Prices for real time kits are at: http://www.twistdx.co.uk/products/twistamp_exo/

If you want to use an RNA template, then you can use this (NB you'll need to add your own RNase Inhibitor at the moment, but we're working on including that too):

http://www.twistdx.co.uk/products/twistamp_rt/

hello everybody,

In my humble opinion, DDPCR is another device that takes the assay planning and assay establishment to the manufacturer. You just take the suitable kit from the supplier, mix it with your material, put it in the device and the software does everything for you. Don´t get me wrong, its fine that scientists have less and less "stupid work" to do. High throughput (ht) techniques are great but very expensive. Not many labs can afford to go on ht, and even, it doesn't make more sense for every application to go on ht. More and more papers and fund proposals have to include ht techniques in order to be accepted, and as a result DDPCR will be the key for future science.

On the other hand, establishing new qPCR assays in the lab is so easy and inexpensive, with the result that it will be the way to go for specific questions, pretrails, and pilot tests.

qPCR cycler are not just used for dna/rna/cdna amplification, but also for methylation analysis and HRM. So, a qPCR device is versatile and covers a wide range of applications.

Conclusion: DDPCR will never replace qPCR. Labs will use both or just the qPCR. Labs without a DDPCR (and of course other ht devices) will outsource this to service supplier.

@MathewForrest That is a great technology improvement. I was checking out your site and am trying to get my PI to order a kit just to try. I like the idea and hope it takes off. I can see where this would be valuable in the field or in underdeveloped parts of the world for a start.

Hi All

I agree with Olivier Switzeny. DDPCR will not completely make normal qPCR obsolete. In ecological studies there is usually at least hundred samples, inhibition may occur (tweaking & re-runs), and post PCR analysis is required (melt curve analysis is minimum).

However, DDPCR is superior over conventional qPCR when accuracy is required for example in RT-qPCR of mRNA template.

Depending on the application, ddPCR will and will not replace PCR. ddPCR is an absolute quantification method with higher accuracy for low abundant targets.

So, for gene expression analysis of regular tissues or cells, I don't see why ddPCR should replace qPCR as it will involve a lot of sample handling, and provide the same results.

However, ddPCR is an improved technology for absolute quantification, for estimating copy number variation and for analyzing samples with low abundant target molecules or variable reaction efficiency.

About the software: Researchers still can extract raw data and analyse it for themeselves (assay planning and establishment is thus not taken to the manufacturer!). It is highly likely that the research community will provide improved software tools in the future, as happend for qPCR normalization.

I agree with ward ddPCR cannot fully replace pcr though it s advance and helps to get result in minimum time period,

We recently purchased QX100, ddPCR. It looks promising than qPCR. Still I agree with most other replies: both will be used for few years to come, as ddPCR technology is still new ... and kits are bit expensive. But for sure it holds the key for the future experiments ...

Not too many applications require high precision of ddPCR, e.g., "normal" gene expression levels vary by ~20% between night and day, males and females, etc. CNVs change from 2 to3 copies can be measured by qPCR; ddPCR can distinguish 5 from 6 copies, but, again 5 or 6 usually does not matter from biology perspective as both indicate expansion of a locus from normal 2 copies. Two big applications for ddPCR are (1) rare somatic mutation detection and (2) fetal aneploidy testing, e.g., trisomy 21. The fist benefits from sample splitting; the second, indeed, requires very high precision. The bottom line, in my opinion ddPCR would not replace qPCR with its installed base of >80,000 instruments..

If DDPCR is as difficult to optimize as the emulsion PCR for NGS then I don't think that it will ever replace real time qPCR technology. This reminds me of the ABI attempt a few years ago to force their 7500 FAST PCR machines to use a two step cycle with a 60 Deg C annealing and amplification step. Of course ABI had a nice online system where you could order their pre-designed primer, probe and enzyme mixes.

Like other commentators have said, the difficulties in design and optimisation of DDPCR reactions seems to place the assay development and establishment with the manufacturer, or the very determined and perhaps skilled user.

For many purposes this "centralisation" is not good. In my work on the GR we have something like 20 or 25 qPCRs that we use almost daily, mostly covering different splice variants. I really don't see manufacturers investing the time to setup so many reactions for just one gene............ As @Oliver_Switzeny says, establishing new qPCR assays is very easy and inexpensive and as a result for anything other that the simplest "routine" quantification of a gene old fashioned qPCR will remain. However, for those that know nothing about the gene of interest, and want a quick system they can order online to validate microarray data or some other technique then the increased sensitivity might be an advantage.

I think that it will remain a balance between the supposed increase in sensitivity of DDPCR against the ease of obtaining good quality data from whatever target you want using the primers you want in the region of the gene you want.

DDPCR its only an additional tool to reduce statistical analisys errors and complementarie to qRT-PCR, but at momment not to remplace them..... maybe when the software and hardware take advances probabily remplace but need to wait at least 1.5 years of evolution and test.

Interesting discussion here! I think digital PCR will take off. It does not bring planning to the manufacturer because you can create your own assays, or buy them. With the release of Bio-Rad's new model http://bit.ly/13UN0OE QX200, you will be able to use TaqMan or EvaGreen chemistry (ddPCR has previously been limited to probe chemistry). The ddPCR technology is versatile and goes beyond the limitations of qPCR.

For example:

http://bit.ly/16wvrmT In the case of the Mississippi baby cured of HIV, ddPCR was used to verify the cure.

http://bit.ly/14eAw7D "Droplet Digital™ PCR quantitation of HER2 expression in FFPE breast cancer samples"

http://bit.ly/1aTGBFw Genetically Modified Food Detection

Hi Mourad, Jochen's comments above are an excellent summary of the advantages of ddPCR over qPCR. From a data perspective, it translates to much lower variability in the data and statistically significant results. I agree with Jochen that ddPCR will eventually replace qPCR because it eliminates many of the pain points of qPCR. Since reaction efficiency is much less of a factor to consider with ddPCR, multiplexing targets in the same reaction is much easier which not only permits better precision because targets are being assayed from the identical sample but it also reduces the cost per target per sample and the amount of precious sample required for a given test. Not only can you multiplex with TaqMan probes but also with intercalating dye chemistry with ddPCR. Finally since ddPCR is an absolute and independant analysis of the DNA amount in each sample, you can initially run technical singlicates and then run duplicates if needed on another day and the data will be highly reproducible which would be challenging with qPCR because of interplate and interassay variablity. Taking all of these factors into account, ddPCR is already very close in cost to qPCR if you factor the technical replicates required for qPCR and the fact that you can easily run duplexed reactions in ddPCR. Even tetraplexing with TaqMan probes is not too challenging.

Just a note of clarification, I am talking specifically about ddPCR from Bio-Rad and the QX200. If you wish to learn more, contact your local Bio-Rad rep for a demo if you are not already testing the technology. I just had an article accepted in the Journal of Virological Methods which will be well worth reading because it deals with this topic and it will hopefully be out in the next few weeks. Go digital! 

Dear Sean 

To talk about the posible things you theoretically can do, is not what users need.

Please show examples