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

Recombinant DNA

Recombinant DNA

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...

RACE - Rapid Amplification of cDNA Ends

RACE - Rapid Amplification of cDNA Ends

Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
Since the...

You might also read

Related Articles

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

Sort by

Same author

A Scoping Review of Nonverbal Synchrony Between Individuals With Social Anxiety and Others.

Psychological reports·2026

Same author

Dianionic Tetraalkynylmagnesate Complexes: A Strategy for Propargyl Amine Synthesis.

Organic letters·2026

Same author

Conversion to Resectional Roux-en-Y Gastric Bypass for Refractory GERD With Predominant Non-Acid Reflux After Single-Anastomosis Duodenojejunal Bypass Diagnosed by Multichannel Intraluminal Impedance-pH Monitoring.

Asian journal of endoscopic surgery·2026

Same author

Prognostic significance of inflammatory cytokine polymorphisms in AML: a study of IFNG, TNFA, and IL1B polymorphisms.

BMC immunology·2026

Same author

Living-donor, single-lobe lung retransplantation combined with lung volume reduction surgery in a patient with emphysema.

JTCVS techniques·2026

Same author

Facile Synthesis of α-Silyl Alcohols via Addition of Trimethylsilyllithium to Carbonyl Compounds.

Chemical & pharmaceutical bulletin·2026

Search research articles

Related Experiment Video

Updated: May 30, 2026

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Published on: February 5, 2021

Self-assembly cloning: a rapid construction method for recombinant molecules from multiple fragments.

Akira Matsumoto¹, Taichi Q Itoh

¹Department of Biology, Juntendo University School of Medicine, Inzai, Chiba, Japan. akirarcb@sakura.juntendo.ac.jp

|July 26, 2011

Summary

Enzyme-free cloning (EFC) efficiently assembles DNA fragments. Six-base pair overhangs with 50% GC content enable one-step construction of large plasmids over 10 kb.

More Related Videos

CRISPR-based Shuttle Cloning: A High-throughput Cloning Method

CRISPR-based Shuttle Cloning: A High-throughput Cloning Method

Published on: June 13, 2025

Use of In Vivo Assembly for High-efficiency Plasmid Construction

Use of In Vivo Assembly for High-efficiency Plasmid Construction

Published on: February 7, 2025

Related Experiment Videos

Last Updated: May 30, 2026

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Published on: February 5, 2021

CRISPR-based Shuttle Cloning: A High-throughput Cloning Method

CRISPR-based Shuttle Cloning: A High-throughput Cloning Method

Published on: June 13, 2025

Use of In Vivo Assembly for High-efficiency Plasmid Construction

Use of In Vivo Assembly for High-efficiency Plasmid Construction

Published on: February 7, 2025

Area of Science:

Molecular Biology
Synthetic Biology
Biotechnology

Background:

Enzyme-free cloning (EFC) offers a rapid method for creating in-frame fusion genes from multiple DNA fragments.
Optimizing EFC protocols is crucial for its practical application in constructing complex DNA molecules.

Purpose of the Study:

To determine the minimal length and sequence characteristics of complementary staggered overhangs required for efficient multi-fragment self-assembly.
To investigate the size limitations of DNA molecules constructible via EFC.
To refine EFC for the one-step assembly of large, functional plasmids.

Main Methods:

Investigated the role of complementary staggered overhangs in directing the self-assembly of multiple DNA fragments.
Assessed the impact of overhang length and GC content on cloning efficiency.
Determined the maximum size of DNA molecules that can be reliably assembled using EFC.

Main Results:

Six-base pair overhangs with a 50% GC content were found to be sufficient for directing the self-assembly of multiple fragments.
The improved EFC method enabled the efficient, one-step construction of a functional plasmid exceeding 10 kb.
The plasmid contained an in-frame fusion domain constructed from four PCR fragments.

Conclusions:

Optimized overhang design (6 bp, 50% GC) significantly enhances the efficiency of enzyme-free cloning for multi-fragment assembly.
EFC is a viable and efficient method for constructing large, functional DNA molecules, including plasmids over 10 kb.
This improved EFC technique facilitates rapid gene synthesis and genetic engineering applications.