# Efficiency and Fidelity of Site-Directed Mutagenesis with Complementary Primer Pairs

## TL;DR

This study improves a DNA mutagenesis method by using new enzymes and finds that primer design affects mutation success and repeat insertions.

## Contribution

The study identifies a novel molecular mechanism involving primer-induced repeat insertions and improves the QuickChange mutagenesis method using SuperFi II and Q5 polymerases.

## Key findings

- Replacing Pfu with SuperFi II and Q5 polymerases increases mutagenesis efficiency to ~100%.
- Insertions of oligonucleotide repeats at primer sites are frequent in failed mutagenesis attempts.
- Primer pairs with 3′-overhangs reduce insertions and enhance mutagenesis efficiency compared to completely overlapping primers.

## Abstract

What are the main findings?
This study improves the QuickChange site-specific mutagenesis method and makes it faster and more reliable by replacing Pfu with SuperFi II and Q5 DNA polymerases.Analysis of the failed plasmids uncovers frequent insertions of oligonucleotide repeats at the primer sites, thereby identifying a novel molecular mechanism by which partially overlapping primers with 3′-overhangs enhances mutagenesis efficiency to the ideal level of ~100%.

This study improves the QuickChange site-specific mutagenesis method and makes it faster and more reliable by replacing Pfu with SuperFi II and Q5 DNA polymerases.

Analysis of the failed plasmids uncovers frequent insertions of oligonucleotide repeats at the primer sites, thereby identifying a novel molecular mechanism by which partially overlapping primers with 3′-overhangs enhances mutagenesis efficiency to the ideal level of ~100%.

What are the implications?
While still less efficient than P3a and P3b mutagenesis, the improved method is very reliable for engineering point mutations and small insertions or deletions.The frequent insertion of short repeats at the primer sites during site-directed mutagenesis with completely overlapping primers not only points to a new direction on how to improve this and related methods and enhance the mutagenesis efficiency further but also sheds light on how insertional mutations occur in cancer and other diseases.

While still less efficient than P3a and P3b mutagenesis, the improved method is very reliable for engineering point mutations and small insertions or deletions.

The frequent insertion of short repeats at the primer sites during site-directed mutagenesis with completely overlapping primers not only points to a new direction on how to improve this and related methods and enhance the mutagenesis efficiency further but also sheds light on how insertional mutations occur in cancer and other diseases.

Based on PCR with complementary primer pairs and Pfu DNA polymerase, QuickChange site-directed mutagenesis has been widely employed, but its efficiency varies from mutation to mutation. An alternative strategy relies on partially overlapping primer pairs with 3′-overhangs, and this strategy has led to the recent development of P3a and P3b site-directed mutagenesis, in which the use of SuperFi II and Q5 polymerases raises the mutagenesis efficiency to ~100%. It is unclear whether these two DNA polymerases also improve the QuickChange method. Herein, we have evaluated this possibility by engineering 46 mutations on seven expression plasmids, two of which possess extremely GC-rich sequences. As Pfu DNA polymerase is a slow enzyme, its replacement with SuperFi II and Q5 polymerases reduced PCR length. Moreover, the average efficiency for each of the seven plasmids ranged from 48% to 69%, thereby outperforming the original QuickChange method. However, this efficiency is still lower than that from the P3a and P3b methods, supporting the superiority of primer pairs with 3′-overhangs. Analysis of the incorrect plasmids from the improved QuickChange method revealed frequent insertions at primer sites. The insertions were derived from primers and varied from mutation to mutation, with certain sites much more prone to such insertions. In comparison, these insertions occurred at a much lower frequency with the P3a and P3b methods, suggesting that primer pairs with 3′-overhangs enhance mutagenesis efficiency by reducing the likelihood to introduce insertions at primer sites. Thus, this study improves the QuickChange mutagenesis method, supports the superiority of the P3a and P3b methods, and uncovers a novel molecular mechanism by which the efficiency of PCR-based mutagenesis with completely overlapping primer pairs is negatively affected.