The distinction between the two is all to do with enzyme kinetics rather than a real distinction in the points of the cycle. The annealing step is really just about the primers. Extension of the PCR product will take place at the annealing temperature but it will be slower because the optimal temperature for the processivity of taq polymerase is 72 degrees, if you lower the temperature it will simply work a little slower. The reason that 60 degrees is used for real time assays using dual labelled hydrolysis probes (but not if you are using SYBR green generally) is that the 3'-5' exonuclease activity that hydrolyses the probe is most active at 60 degrees. In that situation however you usually design primers to anneal at 60 degrees and have a short amplicon to ensure as high a reaction efficiency as possible. So for a standard PCR you usually have an "extension" step at 72 degrees to speed things up. This was always calculated at 1 minute per Kb but with most taqs and modern thermal cyclers with rapid ramping times this can be cut back further. Then you need to consider the downstream reason for the PCR, if you just want a gel then taq is fine, if you want to clone the product and maintain sequence fidelity- particularly for protein expression then you are better using a proof reading enzyme such as Pfu which is a little slower.

Dear Keegan, I am one who would prefer to keep separate all of the PCR cycle stages and not try to combine annealing and extension . Although the PCR may be successful, in general I think you might endanger the specificity of the amplification by having a sub-optimal annealing temperature, and the productivity if you have a sub-optimal extension. In your case, using a combined annealing and extension at 60°C may simply mean that fewer copies of primers bind and that the Pol enzyme is less efficient than would otherwise be the case. However, if you only want a small amount of PCR DNA for cloning (say), then this should not really be a problem. My feeling is that PCRs either work first time, or there needs to be a lot of effort in finding out which part of the protocol is causing problems - so why save a small amount of time in combining the annealing and extension stages and risk having to do it all again? Regards, Andrew.

Hi Keegan, I prefer to keep the denaturing, annealing and extension as separate steps, unless the difference in temperatures between annealing and extension is less than 5 degree Celsius.

As you might be aware, one of the reasons to use higher annealing temperature is high GC content regions (especially if your template is genomic DNA). Some carry out annealing at higher temperatures (close to the extension temperature) while some add DMSO as an additive.

I once tried a two step approach for a GC rich region (as in the way you've mentioned) which didn't work out. The DMSO procedure worked better for me.

I agree with Andrew Spiers on the further requirement of the amplified product.

Keegan, the bottom line is that either system can work, but it depends on your sequence, primers and the amplification system (enzyme, buffer, etc.) There is no way I know to precdict what will work with a new set of primers. Using a 2 step cycle may be a bit quicker, but it may lose specificity or not work at all. The 2-step cycles are common with some real-time kits and cyclers, but when I used them I had to try various times and temperatues to determine what worked well for my particular primers. In a few cases only a three-step cycle would work, at least among the variations I tried.No one can tell youy what will happen with a new set of primers. If the cycling parameters for the primers are published, they should work, even if there may be a bettter prtocol that the authors did not try. If I am using new primers that I designed with known parameters in a program such as Primer3, I have a standard three step program I try them on first, anhd it works almost all the time. For real-time work where I plan to process many samples, I try other programs and optimize it.Your approach of having a standard cycling program for a first run is pretty reasonable. The end result you are looking for, some product to clone or lots of real-time data, is a more important consideration than an arbitrary decision that 2 or 3 steps is better, especially since you have to do all that optimization yourself.

Hi Keegan, I used some time in the past the two-step PCR, but just in cases in which I had to keep the annealing temperature very high (about 60°C) in order to avoid aspecific amplification. However, in my experience the two step PCR is less efficient than the conventional three step.

Hi Keegan,

In some ways, you've already answered your own question. If you are truly trying to optimize your reaction, as you say, then choosing a 2 or 3 step protocol is not the first decision you must make. Start by proper primer design (as others have mentioned). Then when you get the primers, run some tests (perhaps a gradient assay), to determine the best annealing temperature for the primer set in your hands (depending on the taq system, the instrumentation and even the water in your lab, this can vary significantly from the Tm printed on the primer tube). Then, ask yourself what are you optimizing for. Many people use the term very loosely, not really knowing what they are looking for. For example I work in a diagnostic lab where for some assays, we optimize for specificity at the expense of sensitivity, and other assays optimize for sensitivity at the expense of specificity. This is ALWAYS the tradeoff. Of course we try for a "best combination" of specificity and sensitivity, but there is always a tradeoff. Once you've decided all of this, if you find that the annealing temperature for your assay is around 62-72, you can consider wether a 2 step PCR is appropriate for you.

In my opinion, the only real reason for a 2-step PCR is to save some time. In a diagnostic setting, where thermocyclers are running 24/7, this can be important. Usually, in a university setting, saving 30mins is not always important, especially if your are sacrificing sensitivity or specificity of your assays. Hope this helps.

Hi Keegan,

In some ways, you've already answered your own question. If you are truly trying to optimize your reaction, as you say, then choosing a 2 or 3 step protocol is not the first decision you must make. Start by proper primer design (as others have mentioned). Then when you get the primers, run some tests (perhaps a gradient assay), to determine the best annealing temperature for the primer set in your hands (depending on the taq system, the instrumentation and even the water in your lab, this can vary significantly from the Tm printed on the primer tube). Then, ask yourself what are you optimizing for. Many people use the term very loosely, not really knowing what they are looking for. For example I work in a diagnostic lab where for some assays, we optimize for specificity at the expense of sensitivity, and other assays optimize for sensitivity at the expense of specificity. This is ALWAYS the tradeoff. Of course we try for a "best combination" of specificity and sensitivity, but there is always a tradeoff. Once you've decided all of this, if you find that the annealing temperature for your assay is around 62-72, you can consider wether a 2 step PCR is appropriate for you.

In my opinion, the only real reason for a 2-step PCR is to save some time. In a diagnostic setting, where thermocyclers are running 24/7, this can be important. Usually, in a university setting, saving 30mins is not always important, especially if your are sacrificing sensitivity or specificity of your assays. Hope this helps.

Hello,

I believe in what Andrew and Rishikesh mentioned too. Two-step PCR is usually used in real-time PCR with taqman probe.

Best

Dear Keegan,

if you use pre-designed assays, two step-PCRs might be quite common (for example TaqMan probe-based PCR: http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042998.pdf). However, these systems work under standardized conditions (optimized master mix, oligonucleotide-length, etc.). However, if - as mentioned by colleagues before - your oligonucleotides feature appropriate melting temperatures, you could try a two-step approach and compare both methods by amplifying an adequate number of samples under both conditions.

Regards

Dear Keegan,

It is highly important to ensure that the annealing temperature is optimal for your primer set, so therefore I probably wouldn't risk the added complication of a 2-step procedure. Unless there is good reason for optimising a 2-step process, I wouldn't bother reinventing the wheel.

Good luck.

Hello

If you need to optimize your reaction to obtain the best production, I suggest you submit your PCR to a temperature gradient to determine your annealing temperature. In this way you will realize whether to join or not conditions.

Good Luck.

Dear Keegan,

As I feel that Andrew and Mark are correct, it is not a batter choice to mix-up annealing and extension steps in PCR, it can increase nonspecific product if natural annealing temperature for your primer is less than 70.

Hello

Have you already tried a touch-down PCR? It usually works very well!

Good luck with your PCR!

Alessia

I think the question that you should be asking here is for what purpose do you want to optimize your PCR reaction? Speed? Specificity? Quantification as in qPCR? multiplex?

If you use the PCR not for quantification or detection of low amount of template than there is no point on thoroughly optimizing your PCR reaction, just use the general 3-steps PCR protocol and it would be fine.

If you need to thoroughly optimize your PCR reaction, then the only thing to do is to set up an experiment to decide which protocol is the best for your need.

The general rule is 2-steps PCR was intended for speed, as in qPCR and amplification of short PCR prod.

typically I use one step PCR at 60°C for quantitative PCR.

Annealing + extension = 45 seconde to 1 min for 150 pb if you have a polymerase designed for optimal temperature of 72°C.

I tested two step PCR with my primer and I did not find significative difference. May be I obtained a more beautiful melting curve at the end with one step PCR but it's not the case of all my partners.

One major reason why you want to split the anneal/elongation steps is that the elongation step is VERY temperature dependent. So if you were to actually run your reaction with a combined anneal/elongation step at 60° instead of 72° then you'd have to run the step more than twice as long to get the same yield. By running the elongation step at a higher temperture you are maximizing the yield while minimizing the reaction time.

Take a minute to look up the temperature dependance of your enzyme, presumably taq, and decide for youself if you are really helping yourself by combining steps. If you want to go faster and don't care about lower yields then why not just cut your cycles down?

Additionally, the three step process gives you better control over the reation, makes it a lot more forgiving.

Dear colleagues,

To my mind, the "inventor" of the PCR reaction tried to reproduce the natural mechanisms of duplication, accelerating it and reproduce in a short time (x) time (that's no more natural, because in nucleus of cells, it occurs at a certain phase of cycle).

It's a kinetic phenomena (the natural one) and partial duplication can occur at the same time at different part of the chormosome.

Annealing is "the sticking of the primers" , and the linkage is spontaneous by hydrogen bounds. And the temperature for the best sticking depends on the number of A,T,G,C bases of it with 2 or 3 hydrogen bound according to base couple AT or CG). It's the basis of optimisation of the PCR according to composition of primers in bases..

Elongation is active with the action of the enzyme: polymerase, and optimal temperature of elongation depends on kinetic characteristics of the enzyme, especially because thermophilus acquaticus is able (by contrast with our) to "work" at high temperature (so the temperature 70°C ie).

So with one step, it would be possible to take a mean temperature for it , but in this case that's not an "optimisation", because the two steps would be less efficient because not at the optimised temperature for the two processes.

So, thinking at the inital process could allow us to better understand how to optimize our PCR.

Hope usefull.

Best regards

Didier

Fast PCR allows you to combine both annealing and extension steps. Theoretically, this and other modified steps, saves you time from about 2 hrs to as little as 40 min . An enzymes/master mix and Taq polymerase [e,g ampligold] has been optimised by invitrogen for this approach. See invitrogen Fast PCR please.

Hello Dear,

Try to 55-60 oC annealing for better PCR reaction.

Good Luck

Annealing and extension are two separate reactions with different temperature requirements. keep them separate. try to optimize annealing temperature or vary the reaction mixture to optimize your PCR. good luck

Annealing and extension in one step is preferable when the melting temperature of both primers is greater than 72 C. we need to add the time of annealing into the extension time according to length of PCR product. This is called two step PCR. I have tried once this PCR. when I used the general protocol I could not get desired PCR product because melting temp. of primers was more than 72 C, then I went for two step PCR and I got specific PCR product.

As long as I know,if you use a temprature higher than the optimum temprature of your primer annealing they would encounter problem annealing to target sequence.You should use high teprature only if you need specifity in your pcr,so that in these conditions rate of unspecific binding of primers decreases.

Hello,

72°C is the optimal temperature of Taq Polymerase, so :

+if you want a rapid PCR, take 72°C for elongation

+if you choose fidelity (taq is going more slowly but more surely ...), choose 60-68°C elongation step.

It seems that more slowly a tad is going, less mistakes are done and new Taq extend strands at about 60-68°C now. Good Luck, Olivier

shifting annealing temperature is a trade off between specifity and yield. The lower the temparuture, the bigger is yield but the smaller is specifity. The best way how to handle it is gradient pcr, you try differen temperatures and you can see on the gel which one is the best. At low temperatures there will be unspecific products that will be dissapearing until one single band appears and this single band will be fading and fading antil it also vanish. The range of temperatures from "single band appears" to "single band vanish" is the range of annealing temperatures you can use for you PCR.

Both annealing and extension occur throughout the annealing to extension range (50-72°C ). In the normal PCR what is being extended at the 72°C step is actually the primer onto which has already been added a number of bases to make it stable at 72°C. Usually no messing with conditions is required...if it ain't broke!

It has to do with the length of your PCR product. If your product is short (less than 200bp) you can do both steps in once,Otherwise, you have to seperate them.

my addtil. thoughts, but there are so many ways to optimize a PCR reaction, that I only pick some here:

- you may try several annealing temperatures (they differ from the sequence and length of your primers; higher temperatures (in the useful range) in my hands resulted a more specific annealing, but, too high did not give any results.

- if your primers anneal at 60 that should be perfect for your system, so you may play around (i.e. test) some different conditions of annealing time vs. polymerase (72) time.

- one aspect I learned over 20 years ago with PCR (SSCP-PCR and direct radioactive Sequencing) was: not every PCR slot in a metal block really keeps the same temperature, especially when it comes to denaturing... Sometimes half of a degree can be critical...

But I found the first p53 mutation in a medulloblastoma with that technique.

Two-step PCR, where annealing and extension occur at the same temperature is standard in real-time quantitative PCR. The annealing/extension is usually at 60C. This is below the optimum temperature for most thermostable polymerases, which means that extension takes a bit longer. As Ilias points out, if the PCR product is short, this doesn't matter too much. The advantage of a 60C extension step (apart from simplifying the cycling reaction) is that it is easier to design TaqMan probes that will bind to the template strand at this temperature.

For simplicity's sake I design all my quantitative PCRs with a combined extension/annealing at 60C. However, if you're not using TaqMan probes, there's no particular reason to adopt this format.

Agree with Andrew in general.

But sometimes, it depends also on the gene sequence you want to amplify, and the nearest sequences of the gene before and after. Annealing of the primer , when optimized, gives you specificity of your amplification, and elongation and Taq efficiency your performance of amplification (and by contrast: problems if no specificity with primer dimer amplification, or amplification of only a part of your sequence).

But it's also linked to the technic you use: taqman, sybergreen, scorpions probes ..;.

One example: in my experience with factor II gene of coagulation amplification, a part of the sequence of the gene is repeated five times near the exact point to be amplified. So, it wasn't impossible to be specific with the Taqman technic kit from Perkin Elmer, and not efficient; but only with the sybergreen technic (Lightcycler). And in this case, it was really a problem of annealing, because probes couldn't be really specific, whatever the temperature you use for this step. And a fortiori, it was impossible with a one-step PCR.

Regards

Didier

If things work well at separate annealing and extension steps with you, let the thing go as it is. You may prefer a combined annealing and extension step at a particular temp between 60 and 70oC, in case optimizing for development of any diagnostic tool, since a combined step will make method rapid.

The distinction between the two is all to do with enzyme kinetics rather than a real distinction in the points of the cycle. The annealing step is really just about the primers. Extension of the PCR product will take place at the annealing temperature but it will be slower because the optimal temperature for the processivity of taq polymerase is 72 degrees, if you lower the temperature it will simply work a little slower. The reason that 60 degrees is used for real time assays using dual labelled hydrolysis probes (but not if you are using SYBR green generally) is that the 3'-5' exonuclease activity that hydrolyses the probe is most active at 60 degrees. In that situation however you usually design primers to anneal at 60 degrees and have a short amplicon to ensure as high a reaction efficiency as possible. So for a standard PCR you usually have an "extension" step at 72 degrees to speed things up. This was always calculated at 1 minute per Kb but with most taqs and modern thermal cyclers with rapid ramping times this can be cut back further. Then you need to consider the downstream reason for the PCR, if you just want a gel then taq is fine, if you want to clone the product and maintain sequence fidelity- particularly for protein expression then you are better using a proof reading enzyme such as Pfu which is a little slower.

Choosing one or two-step PCR depends on the size of amplicon, Tm of each primer or probe (in condition of using real-time PCR) and the optimum working temperature of your polymerase enzyme.

The best way to determine correct annealing and extension temperature is checking this parameters then decide about steps.

Noel is right. I tried in the past both (60degree for annealing and extension, and 60 for annealing and 72 for extension) in rtPCR and both worked with no apparent dfference. Hence, the 60 degree for both steps saves time. However, does any one know the effect of combining both steps in HRM anaysis? I suppose the HRM dyes are similar to sybergreen rather than to taqman and hence seperate annealing and extension steps?

Khalid makes an interesting statement "with no apparent difference". This is why it is so important to have a properly validated assay. When someone says "no apparent difference", does this mean a few hundread runs were done both ways, analytical sensitivities and specificities were calculated along with calculations of the reaction efficiency for both systems? Because that's what it should mean. Unfortunately, usually when people say this, they mean they ran a few positive controls both ways and they both came out about the same time. I think there is lots of data showing both 2 and 3 step PCRs work, but the choice should be made AFTER determining all the reaction parameters, and knowing what is being gained and lost in each case. If the answer is simply "gained a little time, lost both sensitivity and specificity" then...

Mark, you are right to point out the usefulness of validation using statistical methods. Both approaches had similar CV values and the LoD was not affected. But researchers are encouraged to do their own validation as source of DNA and other parameters could be different.

I prefer 3 step method for real-time PCR analysis, although both 2 and 3 steps works because extension/capture at 72C will increase specificity and reduce chance of primer dimer formation. Regarding annealing temperature, suggest to run a temperature gradient ranging from 55-65C.

Two step PCR is OK as long as you get the sensitivity and specificity right with the right kind of Taq and buffer. If not it is preferable to have the standard 3-step assay where the kinetics is just right.

If the primers are likely to form dimers it is better to use 3 step reaction with extension step in 72 to decrease the time of incubation in low temperatures in which primer dimer formation is more likely. I agree with Bojiang Shen that two step PCR can reduce amplification efficiency, especially if your primers have Tm of 55C - annealing efficiency in, lets say, 60 C would be limiting the amplification efficiency. There is no general advantage of 2 step over 3 step or the other way around. If the primers have low Tm or form dimers I would suggest testing 3 step protocols. With primers that have high Tm two step PCR usually works fine assuring excellent efficiency without reduction in specificity. I perform 2 step PCR with annealing/extension step in 68 C using primers with Tm of around 62 and 65 (calculated by NetPrimer). To my experience the same primers may work fine in two step in one PCR buffer and have specificity issues in another PCR buffer.

Thermal cycler efficiency is also a factor. Some old thermal cycler (100 version)would not perform good for 2 steps reaction. Though it depends on the reagent sensitivity and specificity, most of the time annealing temp at 55 C and extension at 72C works good.

If the annealing temperature of your primers are no higher than 65 degree, you shouldn't combine the annealing and extension step, and instead, you should do then separated. In my lab, a 30s annealing is used, and 1min/kb plus 30s extension is used.

Ultimately, the several points and explanations already provided by the earlier contributors points to the one important fact that most have come to learn only through experience, and that is that you would have to take a couple of these information and use it to optimize for your experimental needs. I'm not sure there's anything else I can add to the earlier posts. Thanks guys!

usually done separately (f.i. 50/30'';72/45'', depending on the lenght of amplicon, minimum 45'', 1'/kb), you can only join them if the annealing T is higher than 65. At this T, you could do an unique step (f.i. 1') at T 65-70

Two step can work for sure. Protocols that include a high annealing temp probably do not need a separate annealing step. Taq polymerase comes from a bug the lives at high temp. However, the enzyme can work at much lower temps. This is the reason why companies generated "activatable" versions, so that amplification is limited or eliminated while putting the reaction up at room temp. "Activatable" versions include antibody or chemically blocked enzymes that become active at high temp only.

I will prefer separate temperature for your PCR particularly. You told that the primers used by you are optimized at 60 degree and it will be a huge temperature difference as you are using taq polymerase. Another thing is it might be useful if you use Pfu polymerase instead of taq pol as pfu shows maximum efficiency at 68 degree. Actually combining temp is mainly fruitful when the temp difference b/w annealing and extension is below 5 degree.

Annealing and Extension temperature depends upon your primer Tm and the Enzyme used respectively. If primers have a high Tm as high as the optimum temperature for enzyme activity, you can combine the steps. Generally its a rare case, so mostly two steps are carried out at different temperatures. One can combine the temperatures if temperature difference is as low as 2-4 degrees, not more than that.

Else you could try and let us know if it works. All d best

Hi Keegan,

Please tell us about your attempts result>>

Good luck

Thanks for all the advice! We tried both, and found little difference. The faculty I talked to here at the University seem to prefer a 3-step cycle as opposed to a 2-step, so I've been using the 3-step to suit their conventions. Past work in our lab has had no trouble with these primers in a 3-step, annealing at 55. Our real-time assays include melt-curve analysis, however, and these aren't as pretty as I'd like - indicating at best the presence of a secondary product, and possibly a primer dimer. But, when we run the product on an agarose gel, we see just one band. It's vexing, but the problem seems to be isolated to the melt-curves - the amplifications and gels are behaving as we expect, and don't seem to affected by the use of separate annealing or extension steps. I'm wondering if maybe there are different versions of our amplicon between different nuclei of our fungi, and those changes in base sequence are what are giving us multiple peaks on the melt-curve (if they changed in length, we'd see that on the agarose gel - but not if they are only changes in sequence). Sequencing or DGGE could probably answer that question.

Dear Keegan,

You have experimented, I thnk, different steps we all met when we put in order a new technic. Remember thta the agarose gel hasn't sufficient resolution, in term of molecular weight , to distinguish two vey near compounds. So, it's not necessary discordant with your two precedent peaks. The only way to distinguish is sequencing of the PCR ampflified products.

Sometimes, even with "specific" reagents, that's not possible to distinguish amplification of near sequences with repetition of similar nucleotidic sequence (ie AAT TGCG AAT gcta AAT..). The same in our lab fgor amplification of coagulation factor II gene with classical PCR on Perkin Elmer.

To my mind, even if it's more time spent to finalise, when there is some difficulties, 3 step PCR could be more "resolutive" than 2 steps. But perhaps am I from a too old generation :-).

Best regards

Didir