For site directed mutagenesis primer design, specifically those designed to make single-nucleotide substitutions, I have seen methods that utilize back-to-back primer pairs that anneal at the 5' ends via phosphorylation and subsequent ligation; as well as methods that use primer pairs with overlapping sequences on their 5' ends. I am curious if there are any significant differences in outcome between these two designs and what, if any, advantages/disadvantages are associated with either design?
I had more effective results using back to back sequences, than overhang. Just use ~16-18 bp exact complimentary sequences, both ways flanking the site to be mutated.
I prefer the design of 5'-phosphorylated primers, one of them is 100% homologous and the longer one will have the mismatch. Inverse PCR: Along with Pfu, next is ligation, then Dpn1. I prefer this method because I can outline the steps, it works everytime. Very reliable.
Oscar L. Sierra why do you prefer 5' phosphorylated primers? Can you please explain it?
I am losing 1 base from the 5' end of the primer when I do insertion mutagenesis, it happened consistently, I start to think it might be degraded.
Interestingly, I didn't lost any base in the reverse primer.
Ahmet Alptekin - Hi, I saw your other post as well. I'm sorry you had this T missing in your mutagenesis experiment. Because the changes looks consistent I would assume is not a sequencing error although it's always good to get an unequivocal reading. -
Using the Q5 from NEB is pretty much the same with my approach of using custom made 5' phosphorylation. In Q5 you use KLD (kinase, ligase, Dpn1). In my case I don't deal with kinases, the primers are ordered, at a larger mass, are more expensive, but usually I request SDS-PAGE purification or the best purification method they have (FPLC once), the amount of primer you receive is much less than what you ordered because the amount will decrease in the purification step but whatever you receive is likely to be correct.
So I do the inverse PCR with a high fidelity polymerase like Pfu, purify, ligate, Dpn1. Transformation. It's usually very robust.
In your case, since you already have the Q5 kit, I would repeat the experiment but reorder the primers with a purification step, it will be more expensive but not as expensive as if asking for a 5'-phosphorylation. It should work because the primers are bound to be correct. But other options are good too. Good luck.
Thank you Oscar L. Sierra . I got the same advice from NEB company too, they suggested purified primers. I'll try that. I appreciate your answer.
Ahmet Alptekin Hi. Good to hear.
The reason for ordering a 5'- phosphorylation is that anyway one of the primers (the one containing the mismatch) is a long primer, which can contain errors introduced during synthesis.
According to this paper, "Nature has evolved sophisticated error-correction mechanisms to ensure that DNA replication proceeds with high fidelity [4]. The error rates in prokaryotic and eukaryotic replication machineries range from 10−7 to 10−8 thanks to various proofreading and mismatch repair mechanisms [5, 6]. In contrast, current gene synthesis process has a typical error rate of 10−2 to 10−3, or 1–10 errors per kilo base-pairs (kbp) synthesized [7–10]."
So in order to avoid erroneous synthesis in a long primer (usually 30-35 bases), it's best to have it purified (bad primers are removed). So having this primer phosphorylated, makes easy the decision to do the same for the other primer (100% homologous) and skip the kinase step. With time, you stop using kits and use more often individual reagents that can be economical in the long run.
Thanks for sharing your experience.
Article Error Correction in Gene Synthesis Technology
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