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Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

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C2CAplus: A One-Pot Isothermal Circle-to-Circle DNA Amplification System.

Laura Grasemann¹, Paula Thiel Pizarro¹, Sebastian J Maerkl¹

¹Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.

ACS Synthetic Biology

|September 20, 2023

Summary

Circle-to-circle amplification (C2CA) was improved to C2CAplus, a one-pot, isothermal method. This DNA amplification technique simplifies procedures and significantly increases DNA yield for easier detection.

Keywords:

DNA replication cell-free synthetic biology circle-to-circle amplification phi29 rolling circle amplification synthetic biology

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Last Updated: Jul 16, 2025

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Published on: October 5, 2018

Area of Science:

Molecular Biology
Biotechnology
Biochemistry

Background:

Rolling circle amplification (RCA) amplifies circular DNA into multimeric linear DNA.
Circle-to-circle amplification (C2CA) enhances RCA by recircularizing products, increasing yield.
Traditional C2CA is multistep and nonisothermal, limiting its practical advantages.

Purpose of the Study:

To develop a simplified, one-pot, isothermal Circle-to-circle amplification method.
To enhance DNA amplification yield and ease of detection.
To overcome the limitations of traditional multistep C2CA.

Main Methods:

Developed C2CAplus, a single-step, isothermal reaction.
Optimized reaction conditions for temperatures between 25-37 °C.
Utilized circular DNA templates for amplification.

Main Results:

C2CAplus functions as a one-pot, isothermal reaction.
The method is robust and simplifies fluid handling.
Achieved high DNA amplification yields, with products visible to the naked eye.

Conclusions:

C2CAplus offers a significant improvement over traditional C2CA.
The simplified, isothermal nature enhances RCA's advantages.
This method provides a simple and efficient way to generate large amounts of DNA product.