What is PCR site-directed mutagenesis?
Site-directed mutagenesis is a molecular biology method that is used to make specific changes to the DNA sequence of a gene. In Lasergene, this workflow utilizes both protein structure and sequence data to produce the best results and save time over traditional trial-and-error methods. If a protein structure is available, start with Protean 3D to scan for hot spots and test the impact of specific variants on the protein fold stability. Next, use SeqBuilder Pro to introduce variants on the sequence, create a mutated primer, and use the primer to perform in silico cloning.
Can I use protein structure prediction to improve my PCR site-directed mutagenesis results?
Yes, Protean 3D provides protein design capability so that you can introduce specific variants to see how they impact protein structures, including the fold stability and developability of the variant structures.
How can I predict folding hot spots in my protein structure?
Protean 3D can predict which residues contribute to fold stability more significantly than others using computational alanine scanning or serine scanning. The more destabilizing the alanine/serine variant, the greater the probability that the position and residue identity is important to the fold.
To predict folding hot spots, choose Modeling > Protein Design > Scan for Hot Spots With > Document. Keep all defaults and choose Run. In most cases, the results of the serine or alanine scan will appear in the Report view in well under a minute.
Can I manually create variants for my protein structure?
Yes. Launch Protean 3D and choose Modeling > Protein Design > Create Variant With > Document. Then use the Substitute drop-down menus to select a different amino acid for each position of interest and press Run. In most cases, results are available in a matter of seconds.
Is there a way to introduce mutations when doing primer design for site directed mutagenesis?
Yes. SeqBuilder Pro’s “Primer Design View” lets you introduce mutations into primers manually or by replacing any codon in the primer with another codon of your choice. The protein translation display will update instantly to reflect your changes. These site directed mutagenesis primers can then be used in the cloning step (see below).
What multi-fragment PCR based cloning options are available for PCR site-directed mutagenesis?
Any of SeqBuilder Pro’s wizard-guided PCR based cloning methods (e.g., Gibson assembly, In-Fusion, GeneArt) and Multi-Site Gateway cloning allow you to easily clone multiple fragments.
Can I perform site-directed mutagenesis without a protein structure?
Yes, simply start the workflow at the primer design step within SeqBuilder Pro, then continue with molecular cloning. While protein structure data is useful for guiding hypotheses for PCR site-directed mutagenesis, it is not required.
