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I've been told that the maximum number of cycles in PCR is between 20 and 30.

Is this true, and what are the reasons for this limitation?

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3 Answers 3

up vote 10 down vote accepted

I would draw the line beyond 35, but thats a bit cosmetic. The reasons are manyfold:

  • due to the exponential fashion of the amplification (ideally) reagents are used up at some point
  • reagents degrade, this is especially true for the dNTPs
  • the activity of the enzyme, despite being heat-stable is declining over time
  • beyond 35 cycles the exponential curve is flattening out (reasons see above)
  • if you run the PCR for too long, you will get more and more side-products (mostly primer dimers, but mis-aligned primers can also make problems), this is more a problem for real-time PCR than for ones run on a gel

If you need a higher sensitivity with more cycles, you can use the technique called "nested PCR". There you do a first round with a primer pair specific for the region of interest and then do a second round with primers which are located slightly to the inside of the amplified DNA. This is done to avoid the amplification of unwanted contaminations. Since you do some 50-70 rounds of PCR amplification in total, this method is extremely sensitive (also to contaminations). See the image from the Wikipedia article for details:

enter image description here

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Thanks for the answer! Would it then be possible to terminate the PCR, isolate out all the DNA and then start a new batch in PCR with all the isolated DNA, as to renew the polymerase and have "new" primers? –  tschoppi Feb 14 '14 at 17:01
Yes, thats possible and is done. But to avoid the amplification of a contamination, you use a set of primers which is located slightly to the inside of the product and which does not overlap with the primers of the first round. This technique is called nested PCR (the article also contains an image of the technique. –  Chris Feb 14 '14 at 17:14
Awesome, thanks for the info! –  tschoppi Feb 14 '14 at 17:19
I have added it to the answer. –  Chris Feb 14 '14 at 17:23

How many cycles of PCR before dNTPs run out?

Assume a 25 μl reaction.

Assume 200 μM dNTPs.

200 μM dNTPs = 200 pmol μl -1

so in 25 μl reaction, there are 5000 pmol of dNTPs

= 5000 x 10-12 x 6 x 1023 molecules

= 3 x 1015 molecules dNTP

Assume that we start with 1 molecule of a 1000 bp template, 50% GC

1 kb = 2000 nucleotides

So , how many of these molecules can we construct using 3 x 1015 molecules dNTP?

= 3 x 1015/2000

= 1.5 x 1012 DNA molecules

How many cycles of PCR to produce this many from a single template molecule?

2n = 1.5x 1012

nlog2 = log(1.5) + log(1012)

nlog2 = 12.18

n = 12.18/ log2 = 41 cycles

Of course this in an absolute upper limit. The estimate assumes that you start with one template molecule of 1 kb, and that dNTPs aren't being hydrolysed, or otherwise degraded.

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You have to take into account, that we use four different dNTPs for PCR, so this calculation would only be valid if the bases are equally distributed. –  Chris Feb 14 '14 at 18:44
Which is why I stated the assumption of 50% GC (and so 50% AT). –  Alan Boyd Feb 14 '14 at 21:27
You are right, I missed that point. Sorry for that. –  Chris Feb 14 '14 at 21:37

In my own research I've found that the amount of PCR product increased as I increased the cycles to 50 times. This was for a single pair of primers used to amplify genomic DNA extracted with a particular kit as doesn't apply to other reactions I've done. What some people seem to forget is that the PCR products don't necessarily double at each cycle at any point in the PCR because some primers don't function as well as others and reaction conditions may be suboptimal (e.g. presence of contaminants or concentration of Mg ions).

In short, provided you aren't getting reduced-quality products (e.g. smearing or non-specific priming products), then I think it's fine to increase the number of cycles well beyond 30 - it's something you have to test empirically with your particular primers, template, polymerase, buffer etc.

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