Traditional Approaches to Site-Directed Mutagenesis
Inverse PCR
For deletion or insertions of >50 bp, inverse PCR is the most popular approach. Inverse PCR uses back-to-back primers to amplify the whole plasmid, followed by ligation of the linear product forming circular DNA. This technique is also suitable for larger insertions or deletions, e.g. removing a regulatory domain from a protein. For deletions, the selected area can be removed by designing primers that anneal at either side of the targeted deletion zone. Amplification proceeds outwards from this area, thus excluding this region from the PCR product. Once the linear product is ligated, your new construct will be lacking this deletion domain. This process is outlined schematically in Figure 1 below.
Figure 1. Protocol for inverse PCR in SDM.
Insertions can be achieved by using overlapping primers with flanking sequences that contain the appropriate extra bases. For more information on inverse PCR check out these resources:- Ochman et al. (1998) Genetic Applications of an Inverse Polymerase Chain Reaction. Genetics. 120:621-623.
- Clifford N. Dominy and David W. Andrews. Site-Directed Mutagenesis by Inverse PCR.
SDM Using Modified Primers
This technique uses modified primers to incorporate small base pair changes into a plasmid, and is the method of choice for site-specific mutations. To start with, you’ll need to design some primers. But before you start:- Print out a copy of an amino acid codon table
- Have your plasmid or protein sequence at the ready.
- Make sure you know where the start codon is and in what reading frame your sequence is read.
Primer Design
Aim for SDM primers of approximately 30 bp in length with your mutated site as close to the center as possible. While it is acceptable to make primers a little longer or shorter as required, there should be a minimum of 12 bp either side of your mutated site. If you need help with primer design, https://molbiol-tools.ca/PCR.htm lists some really useful resources to set you on your way.Pick the Right Reagents
Ordinary Taq polymerase just won’t cut it when it comes to SDM – you need to use a proofreading enzyme. There are a variety of commercially available polymerase kits that are up to the job, incorporating a range of features including high fidelity (proofreading capability) and hot start activation. One possibility is Takara’s In-Fusion HD Cloning Plus – an all in 1 solution that includes a high-fidelity polymerase, a PCR purification kit, cloning enzyme, and competent cells for site-directed mutagenesis. Bear in mind that like most lab reagents, many polymerases come with their own pros and cons – generally labs will have historic reasons for picking one over the other and rarely stray from this. While sticking with what works is sensible, there is no harm in reading the literature on other available resources to make sure you have the best reagents for the job!PCR Reaction
The PCR conditions used will vary depending on your choice of kit and polymerase, as well as the primers you have designed and the size of the product. However, the example shown below is a good starting point.Step | Temp (°C) | Time (s) | Cycles |
---|---|---|---|
Denature | 94 | 15 | 18 |
Anneal | 60 | 30 | 18 |
Extension | 72 | 20/kb plasmid | 18 |
Table 1: Example of SDM thermal cycling program
Keeping the number of cycles low will prevent unwanted mutations from occurring. Eighteen cycles should yield a reasonable amount of mutated product without incorporating unwanted mutations. Note that the number of cycles can be altered during troubleshooting if required.