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PCR

PCR

PCR - Polymerase Chain Reaction

PCR - Polymerase Chain Reaction

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.
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Related Experiment Video

Updated: May 8, 2026

Recombineering Homologous Recombination Constructs in Drosophila

Recombineering Homologous Recombination Constructs in Drosophila

Published on: July 13, 2013

Overlap extension PCR cloning.

Anton Bryksin¹, Ichiro Matsumura

¹Department of Biochemistry, Center for Fundamental and Applied Molecular Evolution, Emory University School of Medicine, Atlanta, GA, USA.

Methods in Molecular Biology (Clifton, N.J.)

|September 3, 2013

Summary

This study introduces a novel cloning method for recombinant proteins, enabling easy DNA insertion into plasmids without restriction enzymes or ligase. This technique simplifies molecular cloning for beginners, enhancing protein production efficiency.

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Last Updated: May 8, 2026

Recombineering Homologous Recombination Constructs in Drosophila

Recombineering Homologous Recombination Constructs in Drosophila

Published on: July 13, 2013

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

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Published on: March 10, 2020

Linear Amplification Mediated PCR – Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear Amplification Mediated PCR – Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Published on: June 25, 2014

Area of Science:

Molecular Biology
Biotechnology
Genetic Engineering

Background:

Increasing demand for recombinant proteins necessitates advanced cloning techniques.
Traditional cloning methods often involve restriction endonucleases and T4 DNA ligase, which can be cumbersome.
A simplified, reliable method is needed for efficient DNA cloning, especially for beginners.

Purpose of the Study:

To develop a restriction-ligation-free cloning strategy for DNA inserts into plasmids.
To provide a user-friendly method for molecular cloning applicable to beginners.
To enhance the efficiency and reliability of recombinant protein production through improved cloning.

Main Methods:

Design and synthesis of chimeric primers with plasmid and insert sequences.
Polymerase Chain Reaction (PCR) amplification of DNA inserts using Phusion® DNA polymerase.
Overlap extension PCR using amplified inserts as mega-primers with circular plasmids.
Digestion of original plasmids with Dpn I to eliminate template DNA.
Transformation of competent Escherichia coli cells with the final product.

Main Results:

Successful cloning of DNA inserts into plasmids without restriction enzymes or ligase.
Demonstration of a simplified cloning process suitable for beginners.
Phusion® DNA polymerase used for both amplification and fusion, allowing unified optimization.
Elimination of parental plasmid DNA via Dpn I digestion ensures product purity.

Conclusions:

The developed method offers an efficient and beginner-friendly alternative for DNA cloning.
This technique streamlines the process of generating recombinant proteins.
The unified use of Phusion® DNA polymerase simplifies reaction monitoring and optimization.