This month's question from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) comes from the “DNA and General PCR Methods” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but they do not represent endorsements by, or the opinions of, BioTechniques.
How can I avoid PCR-induced mutations? (Thread 21522)Q When I make constructs with inserts that are greater than 1 kb, I get at least one PCR-induced mutation in my insert. I tried Invitrogen Platinum HiFi Taq and Invitrogen AccuPrime Pfx, which both promise high fidelity, but this did not solve the problem. Can anyone advise me on how to eliminate PCR-induced mutations?
A1 You should not see many errors with those enzymes. Make sure you are using as few cycles as possible, since a randomly introduced error will propagate with each cycle. I usually run 4 identical reactions of 20–22 cycles each and then pool the products for cloning.
A2 Perform a PCR mutagenesis step with the QuikChange Mutagenesis Kit from Stratagene and fix the mutation.
A3 If you only see one point mutation per clone, you probably do have some correct inserts. Just screen a few more clones to find the one without a mutation. Otherwise, look for some convenient restriction sites that will allow you to swap out a piece with a mutation for a correct region from another clone.
A4 dNTPs of poor quality, that were improperly stored, or that were used at the wrong concentration might lead to mutations.
A5 Is your starting template pure plasmid DNA, or was it extracted from an ethidium bromide–stained gel and visualized with UV? You could be starting with a mutated template.
Do you have highly repetitive microsatellite or AT-rich DNA that might cause the polymerase to slip?
The melting temperature of your PCR may also lead to problems. If you get a lot of product, increase the stringency for proper base incorporation by raising the annealing temperature. You could also ensure that each product is the result of a single pass by decreasing the extension time to reduce unintended concatamerization. This is not an issue if you are amplifying linear pieces of DNA.
A6 If you are trying to sub-clone a gene into an expression vector for protein expression and purification purposes, you might want to keep those clones with point mutations. If the mutations are silent, you can use them unless the mutation created a rare codon for your expression system. If the mutation leads to a minor amino acid change that will not alter the protein structure and function, you can start some preliminary experiments while you work on getting the correct clone.
A7 PCR additives can sometimes cause mutations. I have successfully used regular Taq for sub-cloning fragments as large as 2.5 kb without introducing any mutations. I doubt if it is an issue with Taq polymerase, so look into the quality of your additives.
A8 I achieved good results with Phusion (by Finnzymes) from New England Biolabs and also with iProof from Bio-Rad.
What causes small deletions in TOPO cloning? (Thread 20971)Q I cloned 8 PCR products ranging from 1 to 3.6 kb using Invitrogen's pCR8/GW/TOPO kit. In two cases, I got spontaneous deletions at the cloning boundary. The first clone lost 9 bp and the second lost 5 bp.
All PCRs were amplified with Pfu Ultra for 30 cycles, checked for size and cleanliness on a gel, A-tailed with Taq, and then cloned with the kit. The end of the vector at the cloning site in both cases is CGCCCTT, where the final T is an overhang.
Has anyone else had this problem? Were you able to figure out what caused it and how to avoid it?
A1 I had the same problem. I recently moved 5 different inserts from a pEGFP-N1 vector to a pEXP5-CT vector. The latter is an Invitrogen vector for use with TOPO cloning, so it should have been simple. Four of the inserts worked fine, but I had a lot of problems with the fifth.
The PCR product looked perfect in the gel and showed a 1.5-kb band as expected. However, when I cloned it into the TOPO vector, I got plasmids with a wide range of insert sizes from around 50 bp up to 1.5 kb. Only about 10% of them were close to full length and even those had small deletions in every single case. There were a few different deletions, but the most common was an intervening region between 12 bp direct repeats in our gene of interest. It looked almost like an internal recombination, but I can't be sure of what mediated it. I am certain that secondary structure of the PCR product was not the problem since I cloned the same PCR product successfully into two other vectors (pEGFP-N1 and pGEM-T).
To work around this, I eventually found a clone carrying a different small deletion in a location that let me cut out the damaged portion with a double restriction digest. Performing the same double digest on the original pEGFP plasmid excised a “repair” section, which I could then ligate into place and construct the clone I actually wanted.
There aren't any topoisomerase binding sites in the gene sequence, so that doesn't seem to be the problem. I can only conclude that it was some odd interaction between our insert and topoisomerase or the Top10 cell line.
A2 Small deletions can be caused by overexposure to UV light when checking the product size. Try to minimize the viewing time for checking your band.
A3 Are your primers from a reputable source? Primers are synthesized from the 3′ ends, so primers from unreliable sources may be missing bases at the 5′ ends.
A4 The ends of the insert could have been degraded by nonspecific nucleases. That has happened to me even with sticky-end ligations.
