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Updated: Jun 21, 2026
Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems
Published on: June 14, 2021
Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers
Hirokazu Takahashi1, Kimiko Yamamoto, Toshio Ohtani
1NanoBiotechnology Laboratory, Food Engineering Division, National Food Research Institute, National Agriculture and Food Research Organization, Ibaraki, Japan.
Abstract:
The predominant method for DNA cloning is by propagation in biological hosts, but this method has limitations because certain sequences are difficult to clone using any combination of available hosts or vectors. Recently, multiply-primed rolling circle amplification (MPRCA) has been applied to overcome the problems of the DNA cloning via host cells. However, when MPRCA is used to amplify from minute quantities of DNA template, the products are mostly by-product DNA molecules generated by false priming and primer dimer formation. This study demonstrates that MPRCA using random RNA primers[#x02014]instead of DNA primers[#x02014]blocked the synthesis of by-products and succeeded in amplifying one copy of a circular DNA molecule more than 1012-fold to give microgram quantities of amplification product without using submicroliter reaction volumes. Furthermore, a ligation strategy was elaborated to circularize only the desired DNA sequence and eliminate undesired ligation-products. A combination of these methods was able to amplify and ligate a large construct without undesired DNA sequences and at microgram quantities within one day. Therefore, these methods have the possibility to improve DNA cloning techniques that have been restricted by the limitations of PCR methods or by the host cell.
Insights
This study introduces RNA primers for multiply-primed rolling circle amplification (MPRCA), significantly reducing by-products. This method enables efficient amplification and ligation of DNA, overcoming limitations of traditional cloning techniques.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- Traditional DNA cloning via host propagation faces limitations with difficult-to-clone sequences.
- Multiply-primed rolling circle amplification (MPRCA) offers an alternative but can produce by-products from small DNA quantities.
Purpose of the Study:
- To develop an improved MPRCA method for efficient DNA amplification and ligation.
- To overcome by-product formation and limitations of host-cell based cloning.
Main Methods:
- Utilized random RNA primers instead of DNA primers in MPRCA to prevent false priming and primer dimer formation.
- Developed a ligation strategy to exclusively circularize desired DNA sequences, eliminating unwanted ligation products.
- Combined RNA-primed MPRCA with a specific ligation strategy for large construct amplification.
Main Results:
- MPRCA with RNA primers successfully amplified a single DNA copy over 10^12-fold, yielding microgram quantities.
- The method effectively blocked by-product synthesis, producing clean amplification products.
- Successfully amplified and ligated a large DNA construct without undesired sequences within one day.
Conclusions:
- RNA-primed MPRCA combined with a targeted ligation strategy significantly enhances DNA cloning efficiency.
- These advancements offer a powerful alternative to traditional cloning methods, overcoming host cell and PCR limitations.
- The developed technique enables rapid, high-yield amplification and ligation of large DNA constructs.
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