Question

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. I found the answer! Apparently, it is possible to perform a site-directed mutagenesis with back-to-back orientated primers. 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. 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. Just thought i'd clarify for anyone who is reading this. Typically site directed mutagenesis uses two complementary,overlappingprimers 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.

Answer 1

Site-directed mutagenesis (SDM) with the QuickChange mutagenesis kit involves using primers that introduce a specific mutation and bind back-to-back on opposite strands of a cloning vector's DNA. The high-fidelity polymerase replicates the entire plasmid, including the mutation, through PCR amplification, requiring the mutated DNA sequence to be further amplified and cloned for analysis.

Step-by-step explanation:

Site-directed mutagenesis (SDM) is a method used to make specific and intentional changes to the DNA sequence of a gene and its subsequent protein. The QuickChange mutagenesis kit is a popular tool for performing SDM, as it simplifies the process significantly by removing the need for restriction endonuclease digestion or ligation. To begin with, primers are designed to be complementary to the sequence of the gene of interest within a cloning vector. These primers are also designed to introduce the desired mutation.

Designing Primers for SDM

The primers used for QuickChange site-directed mutagenesis typically contain the mutation within their sequence and are complementary to opposite strands of the double-stranded DNA, allowing them to bind back-to-back. As the polymerase extends from the primers during the PCR cycle, it replicates the entire plasmid, including the introduced mutation. The primer's extension results in nicked plasmid DNA, which calls for the subsequent steps of denaturing the template DNA and transformation into a host for propagation.

PCR Amplification in SDM

PCR amplification enables the production of multiple copies of the mutated DNA sequence. This is critical, as the mutation is introduced within the context of a cloning vector, and the subsequent PCR amplification magnifies the number of mutated vectors that can be introduced into bacterial cells for cloning and further analysis. During amplification, the high-fidelity DNA polymerase is vital to prevent additional unintended mutations.