Site Specific Mutagenesis

BioInnovatise Cloning Team

Updated July 8, 2024

Site specific mutagenesis, also referred to as site directed mutagenesis, is a very common molecular biology technique used to make specific and intentional changes to the plasmid DNA sequence of a gene.

This mutagenesis technique allows researchers and scientists to introduce mutations at precise locations within a gene or GOI enabling the further study of specific protein structure and function, gene expression, and other genetic elements. There are several techniques including CRIPSR / Cas9, QuikChange, and oligonucleotide directed that will preform the necessary mutation a researcher is looking for.

At BioInnovatise, we preform all mutations using the specific technique requested by the researcher in order to achieve the mutation they are looking for on a plasmid DNA construct. Learn more about site directed mutagenesis protocols.

Plasmid DNA Construct Diagram

The Types of Mutations That Occur in Site Specific Mutagenesis:

During site specific mutation productions, various types of mutations can be introduced into a DNA sequence. These mutations are specifically designed to investigate the function of genes, proteins, and other genetic elements. The main types of mutations that can be introduced include:

  • Point Mutations or Substitution Mutation
  • Insertions
  • Deletions
  • Combined Mutations
    • Inversion
    • Duplication
  • Conditional Mutations
  • Codon Optimization
  • Site Saturation Mutagenesis (Learn more about site saturation mutagenesis)
  • Non Coding Region Mutations
Mutagenesis Categories: Site Saturation Mutagenesis; Insertion Mutagenesis; Deletion Mutagenesis; Random Mutagenesis; Saturation Mutagenesis

DNA Sequences Before and After Site Specific Mutations (Point/Substitution, Insertion, Deletion), Random Mutagenesis, and Site Saturation

Plasmid DNA Library Construction Using Site Specific Mutagenesis

Plasmid DNA libraries used for various biotechnology applications can be prepared using site specific mutagenesis. This approach is particularly useful in creating a diverse set of genetic variants for screening and studying protein functions, gene regulation, and other biological processes. There are several strategies to create such libraries:

  1. Site Saturation Mutagenesis Libraries:
    • Principle: Introduce random mutations at a specific site within the gene of interest by using degenerate primers during the PCR amplification step. Learn more about site directed mutagenesis primer design.
  2. Error-Prone PCR Libraries:
    • Principle: Introduce random mutations throughout the gene by using a PCR protocol that increases the error rate of the DNA polymerase.
  3. Combinatorial Mutagenesis Libraries:
    • Principle: Introduce multiple mutations at different positions within the gene.
  4. Random Insertion/Deletion Libraries:
    • Principle: Introduce random insertions or deletions within the gene to study the effects on gene function.
  5. CRISPR-Cas9 Mediated Libraries:
    • Principle: Use CRISPR-Cas9 to create targeted double-strand breaks at specific sites, followed by repair with a diverse library of donor templates.
  6. Oligonucleotide-Directed Libraries:
    • Principle: Use synthetic oligonucleotides to introduce mutations at specific sites.

Can The Length of My Plasmid DNA Construct Affect The Potential For a Successful Site Specific Mutation?

Yes! Site saturation mutagenesis becomes more challenging with longer plasmid DNA constructs for several reasons including primer design and synthesis, PCR amplification, cloning and transformation, and screening and verification.

Our cloning team can preform any mutation on a plasmid DNA construct up to 18 kb. However if your plasmid DNA construct is longer than 18 kb or if you are unsure your plasmid DNA construct has the integrity high enough to achieve your desired mutation, contact our team to discuss your project.

Precision medicine research and development progresses everyday, and with it, the need for high-integrity mutant plasmid DNA.

Want to learn more about the latest in mutagenesis? Our colleagues at ScienceDirect, the American Society for Biochemistry and Molecular Biology, and Genetic Engineering and Biotechnology News continuously collect and publish the latest information on genetic mutation research.

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