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Updated: May 8, 2026
07:04
Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli
Published on: February 5, 2019
Site-directed mutagenesis
1Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Methods in Enzymology
|September 10, 2013
Summary
Site-directed mutagenesis uses polymerase chain reaction (PCR) to alter specific DNA sequences in plasmids. This powerful molecular biology technique enables researchers to investigate protein function and structure by precisely modifying amino acids.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Site-directed mutagenesis is a key technique for genetic engineering.
- Understanding protein structure-function relationships is crucial in molecular biology.
Purpose of the Study:
- To explain the principles and applications of site-directed mutagenesis.
- To highlight its utility in studying protein function and interactions.
Main Methods:
- Utilizes polymerase chain reaction (PCR) for targeted DNA modification.
- Involves introducing specific nucleotide changes within a plasmid vector sequence.
Main Results:
- Enables precise alteration of amino acid sequences.
- Facilitates the study of protein structure and function.
- Allows for the creation of modified constructs for further experiments.
Conclusions:
- Site-directed mutagenesis is a versatile tool for molecular and genetic research.
- It is essential for dissecting protein roles and developing novel genetic constructs.
Related Concept Videos
01:16In vitro Mutagenesis
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
01:16In-vitro Mutagenesis
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
02:53Conservative Site-specific Recombination and Phase Variation
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
01:30Spontaneous and Induced Mutations
Spontaneous mutations arise infrequently during DNA replication due to errors in the process. A key factor behind these errors is tautomeric shifts in nitrogenous bases, where bases transition from keto to enol forms or amino to imino forms. This shift can alter base-pairing rules, leading to mutations. Additionally, reactive oxygen species (ROS) arising from aerobic metabolism can damage DNA, resulting in depurination (loss of a purine base) or depyrimidination (loss of a pyrimidine base).