Checking of PCR products?

Question

Does anyone know how to check the quality of the product after a PCR?

As we know TAQ polymerase can make mistake. So, how do we check whether has the Polymerase made an error in between? Also how to prevent the errors from happening. Please help asap.

To add on: Check meaning the sequence that is being PCR is correct. For example, AAATTTATATATGGGCCC would come out as AAATTTATATATGGGCCC and not somewhere in the middle there is a different nucleotide.

THanks for the answers guys, how can gene sequencing be done or is that another method available. I heard of Sanger method but do not know what that is. Can anyone elaborate a little?

Answer 1

Sequencing is your only option, unfortunately. Restriction digest would take you ages and you should test all the known enzymes to see whether the products are identical before and after PCR.

Taq polymerase DOES make mistakes, not at very high rate, but it does. The advantage is that it creates A- overhangs in the product, so that you can clone it into a pET vector.

If you don't need this feature, you could use PfuI, which is much more precise and trusthworthy, but does not create the overhangs. I seem to remember a product called Taq2000, which is a mix of the two polymerases and achieves both high fidelity and ability to clone the product in the pET system.

Answer 2

yeah, sequencing is your best bet. Most facilities can do PCR products, using your PCR primers as sequencing primers. Don't forget to do both forward and back to make sure there are no mismatches. Of course, that will just tell you whether you've probably got relatively clean product or not.
We use the Roche high fidelity kit when accuracy is critical, but there are several high-accuracy enzymes or mixtures of Taq and a proofreading enzyme like Pfu.
If you absolutely must have all correct sequence, the old standard of putting the product into a plasmid PCR vector such as Qiagen's pDrive, transforming a host, cloning and sequencing the individual clones is the way to go.

Answer 3

You either have to sequence it, or you need to assay using a sensitive probe that would detect a single nucleotide difference in the area you are most concerned about.

That's it as far as I know. PCR doesn't generally make a lot of errors though, especially with short stretches.

Answer 4

run on a gel if the pcr product is not what you expect i.e. a mistake has occured there will a discrepency between what you see on the gel and what you expected.... very quick and easy

you could also probe or sequence the product... long and complicated

taq is less problematic than you think keep an eye on your primers