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PCR SITE-DIRECTED MUTAGENESIS

Determine desired variants by scanning for hot-spots and viewing predicted structural changes, then design PCR primers to introduce the variants you identified. PRICING DOWNLOAD FREE TRIAL

See the structural impact of a mutation before designing your primers.

PCR site-directed mutagenesis is one of the most often-used techniques for introducing a mutation into a DNA sequence, however most software tools don’t consider the impact of a mutation on protein structure when designing primers. Lasergene provides a unique approach to primer design for PCR site-directed mutagenesis by first focusing on predicting which mutations introduced into a DNA sequence may lead to changes in protein stability. Using our hot spot scanning and create variant tools, you can quickly see what mutations will increase or decrease fold stability. Once optimal candidate variants for your experiment are determined, you are ready to create PCR primers to introduce them into your sequence using our point-and-click tools for designing and mutating primers. The unique combination of our structure modeling and primer design tools saves you time and money compared to traditional trial-and-error approaches to mutagenesis experiments.

PCR Site Directed Mutagenesis

PCR site-directed mutagenesis in 4 simple steps

PCR Site Directed Mutagenesis Step 1

Step 1

Scan for hot spots

PCR Site Directed Mutagenesis Step 2

Step 2

Create variants in positions of interest

PCR Site Directed Mutagenesis Step 3

Step 3

Create primers to introduce the desired mutations

PCR Site Directed Mutagenesis Step 4

Step 4

Introduce mutated gene into sequence

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Learn more about PCR Site-Directed Mutagenesis

Resources | Tutorials | FAQs | User Guide

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Resources

Please see our resources below for more information on PCR site-directed mutagenesis.

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How to perform PCR site-directed mutagenesis using Lasergene’s protein and primer design tools

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Case Study: Structure-Guided Molecular Cloning

Read Case Study

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Structure-guided molecular cloning for improving site-directed mutagenesis and stability in protein design

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What’s New in Lasergene 16?

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Master Our Foundation Application, SeqBuilder Pro

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SeqBuilder Pro FAQ: Answers to Your Webinar Questions

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Tutorials

Watch one of our videos or check out our user guide to learn more about our PCR site-directed mutagenesis workflow.

Lasergene's Protein Design Workflow

This video shows Lasergene’s Protein Design workflow, which begins in Protean 3D and can optionally finish with primer design and virtual cloning in SeqBuilder Pro.

Primer Design in SeqBuilder Pro

In this video, we show you how to design and modify PCR primers in SeqBuilder Pro.

Virtual Cloning in SeqBuilder Pro

See how to create virtual clones quickly and easily using SeqBuilder Pro.

FAQs

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…

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.

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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…

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.

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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.

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Extremely valuable

“SeqBuilder Pro is easy to use and intuitive. It is also extremely valuable for complex cloning and mutagenesis projects.”

Gilbert Martinez, University of Washington

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