How do nicks in the DNA strand affect the success of Long Range PCR?

Question

Long Range PCR using NEB Master Mix - Hot Start Taq was working fine for me (amplicon sizes of ~10kb) but stopped working all of a sudden. Is it possible that many freeze-thaw cycles on the DNA template introduces nicks into the DNA strand that hamper the polymerase activity ? In certain cases, I am getting no amplification and in some I am getting very weak amplification. Because the integrity of the DNA should be intact over the entire length in case of long range PCR, am I right in thinking nicks in DNA will be especially detrimental if we are trying to amplify large fragments ?

The concentration of the DNA templates I used was 90-140ng/ul in a 10ul PCR reaction, DNA eluted in TE buffer after a magnetic bead method of DNA extraction.

Answer 1

It is not unlikely that nicking is causing trouble. The question is if repeated freezing and thawing is causing the problem, there is an article which has been published in B.R.L. Focus in 1983 which disputes this effect. You can find the PDF with the article here, the article itself starts on page 10 in the PDF. On the other hand there is this paper ("Impact of long-term storage on stability of standard DNA for nucleic acid-based methods."), which shows that sstorage conditions do have an effect on the effciency of qPCR standards. Whatever causes nicking, it is a problem in (long) PCR. The following quote is from Sigma in their long PCR enzyme mix:

Template High quality and adequate length of the template are essential for reliable amplification of larger fragments. Extreme care must be taken in the preparation and handling of the DNA target for long PCR. Nicked or damaged DNA can serve as a potential priming site resulting in high background. Avoid freezing, or, alternatively, freeze only once to minimize damage. The condition of the target DNA is critical. Depurination during cycling is minimized by use of buffers with a pH greater than 9.0 at 25 °C.

This paper ("Effect of Highly Fragmented DNA on PCR") shows that induced nicks affect the efficiency of PCR (although they do a much shorter amplification than you). So to overcome this problem I would make small aliquots of fresh DNA preps and then see, if this helps.