Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers
After the submission of this manuscript, we became aware of a patent describing the use of RNA primers to suppress nonspecific RCA background (37). However, there are two differences between the patent and the present paper. First, the patent has been developed for the detection of single-strand circular probes such as a padlock-probe (11,38); thus, the patent requires RNA primers with long, specific sequences to detect a specific probe. In contrast, the present study used random RNA primers with short sequences to expand the versatility of MPRCA. Second, differences in the length of the RNA primers affect the reaction temperatures used in these two methods. Because accurate annealing of a long, specific RNA primer requires a high temperature, the patented method needs Bst DNA polymerase (large fragment), which works optimally at 65°C. In contrast, random hexamers are unsuitable for reactions at 65°C, and the φ29 DNA polymerase-directed reaction is performed at 30°C. In addition, the fidelity of Bst DNA polymerase is lower than that of φ29 DNA polymerase, because Bst DNA polymerase lacks 3′–5′ exonuclease activity (39). Thus, these differences indicate that combined use of specific RNA primers and Bst DNA polymerase is not suitable for the MPRCA reaction, especially not for cell-free cloning.
In this study, it was demonstrated that cell-free cloning of a ligated, large construct is one of the potential applications of RNA-primed MPRCA. This amplification method can amplify minute quantities of circular DNA: even if only a few copies of ligated circular products are available (due to low ligation efficiency, for example), this method could allow amplification of the desired DNA sequence. Furthermore, provided the number of DNA fragments is the same as the number of kinds of restriction enzyme used, this ligation strategy does not have any limit on the number of DNA fragments that can be used. The amplified DNA product may be suitable to use for the transformation of mammalian cells because the product DNA does not include endotoxins of bacterial origin. Recently, gene-synthesis technology with the elimination of sequence errors—as carried out using oligonucleotides, thermo-stable ligase, plural exonucleases, and endonuclease V—has been reported (40). Although this method may provide great advantages in the chemical synthesis of genes, it has limitations originating from the use of PCR. MPRCA combined with RNA primers may overcome this limitation because the desired product of the method is in a covalently closed circular duplex form.
Circular DNA amplification by MPRCA using RNA primers could greatly improve DNA cloning techniques that are restricted by the limitations of PCR (encountered with highly repetitious or large sequences) or by the host cell being used (encountered with unstable or toxic sequences in host cells) (3,5,6).
The authors thank Jun'ichi Wakayama, Kazumi Tsukamoto, Toshiro Kobori, Yukio Magariyama, and members of the Nanobio-technology group for helpful discussions; and Atsuko Matsumoto, Hiroko Kanahara, and Kanae Tsukada for excellent technical assistance. This work was supported in part by the Bio-Oriented Technology Research Advancement Institution.
The authors declare no competing interests.
Address correspondence to Shigeru Sugiyama, Nano-Biotechnology Laboratory, Food Engineering Division, National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. e-mail: [email protected]
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