I have been performing site-directed mutagenesis with the goal of substituting different amino acids. I have done this successfully three times but when I was attempting my fourth sequencing results came back that there were ~30 unwanted amino acids inserted in my sequence. What could be the cause of this? Could my DNA destabilize while waiting to get picked up and insert other portions of the DNA?
So site directed mutagenesis sounds pretty simple right? Just design a forward primer with a mutated base or two and create a reverse primer that is the exact reverse complement of your forward primer. Polymerase chain reaction with a high fidelity DNA polymerase, dNTPs, magnesium, forward and reverse primers, and your template plasmid DNA. Degrade the parental template DNA and keep the PCR product and transform it into bacteria, extract the DNA and sequence the hopefully mutated plasmid DNA.
So there are two possible products from PCR transformation in cells, not one.
Product 1 that you expect: Your mutated plasmid that codes for a new amino acid at the position(s) you desired.
Product 2 that you don't expect, isn't documented very well, but it is logical:
Your mutated plasmid that codes for a new amino acid at the position(s) you desired ... with the forward or reverse primer inserted to fill the nick just outside of where the primers were.
This is because when site directed mutagenesis PCR works, the PCR product is actually a nicked plasmid that isn't ligated together because there is no DNA ligase. Transformation into E.coli cells ligates the plasmid together ideally at the nick created by the primers. But sometimes E.coli cells see all those excess of primers and use those primers to fill the nick and then ligate the plasmid together.
So the insertion is likely your primer or its complement. If the insertion is in-frame then that is new free construct to study. If the insertion is not in frame then you should throw the extracted DNA away.
Product 2 is less likely to occur if you use a PCR cleanup kit to remove dNTPs and primers from the product of the PCR reaction.
I agree with the second point of Adron. I have had Primer repeats quite frequently in my SDM experiments. Sometimes my primer sequence was integrated >3 times, which could explain your ~30 excess amino acids.
Its very easy to check: Just search for the primer sequence or a excerpt of it in your sequencing results and see if there are multiple hits.
Good luck!
-Jan
Collin Sroge As you have sequencing data: What is the insert? Primers, as suggested by the previous respondents, or sth else? Have you BLASTed the inserted sequence in that case?
- Badges
- Science topic
- Similar topics
- Filing