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Related Concept Videos

Homologous Recombination02:31

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...
Homologous Recombination02:31

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...
Conservative Site-specific Recombination and Phase Variation02:53

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...
Mismatch Repair01:20

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...
Mismatch Repair01:36

Mismatch Repair

Overview
Coordination of Gene Expression Processes in Bacteria01:29

Coordination of Gene Expression Processes in Bacteria

The DNA replication, transcription, and translation processes are intricately coupled in bacteria, allowing efficient gene expression and rapid protein synthesis. While this physical and functional coordination is advantageous, it introduces challenges that bacteria overcome through specific regulatory mechanisms.Coupling of Replication, Transcription, and TranslationThe coupling of replication, transcription, and translation is a hallmark of bacterial gene expression. As the replisome unwinds...

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Related Experiment Video

Updated: Jun 9, 2026

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
09:02

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Published on: January 8, 2015

Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate.

J A Mosberg1, M J Lajoie, G M Church

  • 1Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. jmosberg@fas.harvard.edu

Genetics
|September 4, 2010
PubMed
Summary

The lambda Red recombination system in E. coli uses a novel single-stranded DNA intermediate, not double-stranded, for genetic manipulation. This finding clarifies the mechanism and enhances recombination efficiency.

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Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
07:55

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Published on: September 11, 2022

Related Experiment Videos

Last Updated: Jun 9, 2026

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
09:02

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Published on: January 8, 2015

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
07:55

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Published on: September 11, 2022

Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • The phage lambda-derived Red recombination system is widely used for genetic engineering in Escherichia coli.
  • The precise mechanism of lambda Red-mediated double-stranded DNA recombination remains incompletely understood.
  • Existing models propose a double-stranded DNA intermediate with 3' overhangs generated by lambda exonuclease.

Purpose of the Study:

  • To elucidate the detailed mechanism of lambda Red-mediated recombination.
  • To propose and validate an alternative model involving single-stranded DNA intermediates.
  • To leverage mechanistic insights for improving Red recombination efficiency.

Main Methods:

  • Investigated the recombinogenic potential of single-stranded DNA insertion cassettes.
  • Analyzed strand-specific mutations in double-stranded DNA cassettes.
  • Utilized phosphorothioate linkages to protect specific DNA strands.

Main Results:

  • Single-stranded DNA cassettes were found to be recombinogenic and preferentially targeted the lagging strand.
  • Strand-specific mutations in double-stranded cassettes showed high cosegregation rates.
  • Modification of lagging-targeting strands enhanced lambda Red recombination frequency.

Conclusions:

  • The study proposes a new model where lambda Red recombination proceeds via a single-stranded DNA intermediate.
  • This intermediate is formed by complete strand degradation by lambda exonuclease and subsequent annealing at the replication fork.
  • The findings provide mechanistic insights that can be applied to optimize the lambda Red system for genetic manipulation.