Jump to content

PCR question

Recommended Posts

If you are doing PCR with the intention of sequencing the product and checking a short region of the PCR product for a mutation, is there a region where taq polymerase is more or less likely to make a mistake? For example would it be more likely taq makes a mistake near the primers or in the middle of the PCR product? Should I be using a high fidelity enzyme even if it is only a short (20-25 bp) piece that I am concerned with?

Link to comment
Share on other sites

As a rule of thumb the error rate scales most strongly with the number of reactions (i.e. length and cycles). The lenght is not an issue here, so keeping the cycle number as low as possible can help. Think of it that way, during any single reaction there is a (mostly) stochastic chance of an error, and then there is the chance that this molecule is used as template (and hence increasing erroneous products within the population). Sequence also has an influence, though unless you have runs (i.e. a stretch of identical bases) it is most likely less of an issue. Of course, using high fidelity polymerases are always preferred.

Remember you do not amplify the 25 bp once, but many times and the error rate is a function of the total reactions occurring within the sample.

Link to comment
Share on other sites

  • 2 weeks later...

Create an account or sign in to comment

You need to be a member in order to leave a comment

Create an account

Sign up for a new account in our community. It's easy!

Register a new account

Sign in

Already have an account? Sign in here.

Sign In Now
  • Create New...

Important Information

We have placed cookies on your device to help make this website better. You can adjust your cookie settings, otherwise we'll assume you're okay to continue.