Search research articles

ABOUT JoVE

Overview Leadership Blog JoVE Help Center

AUTHORS

Publishing Process Editorial Board Scope & Policies Peer Review FAQ Submit

LIBRARIANS

Testimonials Subscriptions Access Resources Library Advisory Board FAQ

RESEARCH

JoVE Journal Methods Collections JoVE Encyclopedia of Experiments Archive

EDUCATION

JoVE Core JoVE Business JoVE Science Education JoVE Lab Manual Faculty Resource Center Faculty Site

Terms & Conditions of Use

Related Concept Videos

Conservative Site-specific Recombination and Phase Variation

Conservative 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...

In vitro Mutagenesis

In 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.

Homologous Recombination

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...

In-vitro Mutagenesis

In-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.

Gene Conversion

Gene Conversion

Other than maintaining genome stability via DNA repair, homologous recombination plays an important role in diversifying the genome. In fact, the recombination of sequences forms the molecular basis of genomic evolution. Random and non-random permutations of genomic sequences create a library of new amalgamated sequences. These newly formed genomes can determine the fitness and survival of cells. In bacteria, homologous and non-homologous types of recombination lead to the evolution of new...

Mismatch Repair

Mismatch Repair

Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by

Same author

Glycovariant-based diagnosis of bladder cancer using urinary mucin 1 and carcinoembryonic antigen.

Journal of pharmaceutical and biomedical analysis·2026

Same author

Improving genotyped functional screening: A versatile closed-tube PCR for Illumina library generation reduces background sequences in multiplexed sequencing.

SLAS technology·2026

Same author

Development of an Anti-Immunocomplex Antibody and Non-competitive Immunoassay for the Detection of Testosterone.

Analytical chemistry·2026

Same author

Fluorine-18 Radiolabeled Single-Chain Antibody Variable Fragment 1F4 Targets α1-Subunit Gamma-Aminobutyric Acid Type A Receptors in Mice.

Journal of medicinal chemistry·2026

Same author

Corrigendum to "Aberrantly glycosylated PSMA in urine as a potential marker for prostate cancer" [Clin. Chim. Acta 582 (2026) 120790].

Clinica chimica acta; international journal of clinical chemistry·2026

Same author

Aberrantly glycosylated PSMA in urine as a potential marker for prostate cancer.

Clinica chimica acta; international journal of clinical chemistry·2025

Search research articles

Related Experiment Video

Updated: Jun 3, 2026

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Published on: November 20, 2016

Enhanced error-prone RCA mutagenesis by concatemer resolution.

Tuomas Huovinen¹, Marja Julin, Hanna Sanmark

¹Department of Biochemistry and Food Chemistry, University of Turku, Tykistökatu 6A, 6th floor, 20520 Turku, Finland. Tuomas.Huovinen@utu.fi

|April 2, 2011

Summary

This summary is machine-generated.

Error-prone rolling circle amplification (RCA) can now generate larger mutant libraries. Introducing Cre/loxP recombination significantly boosts transformation efficiency, making RCA a more powerful tool for random mutagenesis and protein engineering.

More Related Videos

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Published on: August 25, 2017

Related Experiment Videos

Last Updated: Jun 3, 2026

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Published on: November 20, 2016

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Published on: August 25, 2017

Area of Science:

Molecular Biology
Biotechnology
Genetic Engineering

Background:

Error-prone rolling circle amplification (RCA) is a method for generating random mutations.
A key limitation of RCA is the low transformation efficiency of amplified DNA concatemers.
This hinders its utility for creating large mutant libraries.

Purpose of the Study:

To enhance the transformation efficiency of error-prone RCA.
To improve the generation of diverse mutant libraries for protein engineering.

Main Methods:

Incorporated bacteriophage P1 Cre recombinase loxP sites into the target plasmid.
Utilized Cre recombinase to reduce RCA-generated DNA concatemers into plasmid-sized units.
Assessed transformation efficiency with and without Cre/loxP recombination under error-prone and non-error-prone conditions.

Main Results:

Cre/loxP recombination increased transformant numbers 50-fold under non-error-prone conditions and 13-fold under error-prone conditions.
A library of randomly mutated TEM-1 β-lactamase genes yielded 115 ± 57 resistant colonies per recombined RCA reaction, compared to only 9 ± 11 without recombination.
Demonstrated a significant improvement in library size and diversity.

Conclusions:

Supplementing error-prone RCA with Cre/loxP recombination is an effective strategy.
This method substantially increases the size of transformable mutant libraries.
It offers a simple and valuable tool for random mutagenesis and directed evolution.