4
$\begingroup$

In our practical course about modern cloning methods, we performed point mutations on a promotor via site-directed mutagenesis. As far as I understand that method you need forward and reverse primers with a partial overlapping sequence to perform point mutations. For deletion and insertion you need forward and reverse primers with a back-to-back orientation. In the practical course, we already performed that step of the mutation. However, after taking a closer look via CLC workbench where we performed that reaction in silico, I saw that our used primers had no overlapping sequences. Instead our forward and reverse primers had a back-to-back orientation. Our mutation did work out.

Therefore, I was thinking that point mutation via SDM is also possible with back-to-back primers. However, I did not find any source that can prove that theory of mine. So I am hoping that someone with more experience than I have, could help me out.

$\endgroup$
3
  • 2
    $\begingroup$ It would be useful it you could make an example drawing/figure to explain what you mean. I'm currently having a hard time understanding your question. $\endgroup$
    – gaspanic
    Commented Jul 17, 2022 at 14:27
  • $\begingroup$ Thank you for your comment! I now edited my question and included some screenshots of my analysis in the CLC workbench. Even though I found the solution now, do you understand what I meant? $\endgroup$
    – Natalie
    Commented Jul 17, 2022 at 17:07
  • 2
    $\begingroup$ Yes, I understand now. Since you have no primer overlap, you probably used 5' phosphorylated primers, amplified the entire plasmid, and then ligated the resulting PCR product to create the new, mutated plasmid. You might want to remove your edit from the question and instead post it as an answer. Also, you did not perform permutations, you inserted mutations. Permutations is something different. $\endgroup$
    – gaspanic
    Commented Jul 17, 2022 at 18:14

2 Answers 2

2
$\begingroup$

I found the answer! Apparently, it is possible to perform a site-directed mutagenesis with back-to-back orientated primers.

WT Plasmid

In that picture, the green mark represents a part of the lacZ gene. There, we performed a point mutation by using the forward primer "Cand2 M5 Fwd" and the reverse primer "Cand2 M5 Rev". The forward primer is in fact longer: it includes the purple highlighted part as well. There we exchanged some nucleotides.

Mutated Plasmid

In this picture, some nucleotides in the once highlighted part are now exchanged. I was confused as I was tought that the primers should have a partial overlapping sequence with each other. But now as they are back-to-back orientated and have no matching sequence with eacht other, I discoverd that it is possible to perform point mutations like this in a template sequence.

$\endgroup$
5
  • 1
    $\begingroup$ This doesn't really answer your question though. Like I pointed out in my comment to your question, my guess is that you used phosphorylated primers and then went through a ligation step to seal the plasmid before transformation into bacteria. But whether that's what you actually did or not is not clear from your answer. $\endgroup$
    – gaspanic
    Commented Jul 17, 2022 at 19:48
  • $\begingroup$ Hello there! Maybe you understand something wrong. We indeed used back-to-back primer which were phosphorylated and then ligated the plasmid with the wanted mutations. However, with my question I was focusing on the POSSIBILITY to use back-to-back primers for point mutations. Maybe I misunderstood that in class, but I thought you have to use overlapping primers. Therefore, I was very confused as I saw our in silico experiment and reached out to get an explanation from more experienced people (my prof didn't answer my mail...). $\endgroup$
    – Natalie
    Commented Jul 19, 2022 at 13:41
  • 1
    $\begingroup$ Got it. Thing is that in traditional site-directed mutagenesis (with homologous primers) you don't use phosphorylated primers, nor do you depend on a ligation step. Two completely different methods. $\endgroup$
    – gaspanic
    Commented Jul 19, 2022 at 14:27
  • $\begingroup$ Hi there! Thank your for your response. Do you mind telling me more about this? Is there a possibility to write something like a private message? $\endgroup$
    – Natalie
    Commented Jul 21, 2022 at 8:05
  • 1
    $\begingroup$ There is not, but you can look for the QuikChange method. $\endgroup$
    – gaspanic
    Commented Jul 21, 2022 at 13:43
2
$\begingroup$

Just thought i'd clarify for anyone who is reading this.

enter image description here

Typically site directed mutagenesis uses two complementary, overlapping primers which each contain the mutation desired in the plasmid. then PCR will generate the entire plasmid.

There is a second method with only one primer for each desired mutation. The primers are phosphorylated by a kinase enzyme to make the primers work to accept ligation.

This requires a high fidelity polymerase because PCR is copying the entire plasmid else unwanted additional mutations will occur. This sounds simpler but you have to sequence the mutation sites of several colonies to get full results.

$\endgroup$

You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .