molecular biology - Why is the number of PCR cycles limited?

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?

Answer

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: