2Insect Genome Laboratory, National Institute of Agrobiological Sciences, Ibaraki, Japan
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—instead of DNA primers—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.
The most popular method for cloning foreign DNA is still via propagation in vectors—predominantly plasmids—using biological hosts such as Escherichia coli, followed by their isolation. However, these methods have limitations, because many sequences are difficult to clone using any combination of available hosts or vectors. For example, retroviral long-terminal repeats are not maintained stably in host cells (1), and large constructs, such as gene-targeting vectors to produce knockout mice, frequently show low transformation efficiencies (2).
PCR is a common method used in the cell-free cloning of DNA molecules. However, PCR is not suitable to amplify highly repetitious or large sequences. For these reasons, a new cell-free enzymatic system for cloning difficult DNA sequences has long been desired. Recently, Hutchison and colleagues reported a novel cell-free cloning method using multiply-primed rolling circle amplification (MPRCA), which can amplify individual DNA molecules using submicroliter reaction volumes (3). This method provides one way to overcome the problems of foreign DNA cloning via host cells, because MPRCA, which is based on rolling circle amplification (RCA), has little limitation in terms of the amplification length or sequence (3,4,5,6). MPRCA has also been applied to the amplification for circular DNA directly from ligation reactions, including cDNA libraries (5,7).
MPRCA was developed using bacterio-phage φ29 DNA polymerase and protected random DNA primers as a method to amplify circular DNA molecules (8). Bacteriophage φ29 DNA polymerase has exceptional processing, high proofreading, and efficient strand displacement activities (9,10,11); thus, the amplified product is useful for many downstream analyses (4,5,7,8)(12,13,14,15,16,17,18,19,20,21). The crucial point of this method is the use of random DNA primers; thereby, this method can amplify the target DNA without prior sequence information, such as in the amplification of a novel circular DNA virus (14,15). However, the use of random DNA primers contributes to a critical problem with MPRCA. When MPRCA is used to amplify minute quantities of DNA template, downstream analyses are limited because most of the resulting molecules are by-products generated from undesired primer-directed DNA synthesis (22,23). These by-products are also found in negative control samples without adding template DNA (22). Therefore, it is necessary to judge the success or failure of the amplification by other analyses such as restriction enzyme digestion, sequencing, or PCR amplification of specific regions (3,18)(24,25,26). Although optimized reaction conditions, including modified primers, have been reported to reduce the levels of by-products (22,23,24)(27), these modified methods were almost completely ineffective in increasing the yield of desired DNA product via long reaction periods (24,27).
Here, we report that random RNA primers can block the synthesis of by-products derived from primers in the MPRCA reaction. Bacteriophage φ29 DNA polymerase can use RNA as a primer for DNA synthesis (28), but cannot use RNA as a template because φ29 DNA polymerase is a DNA-dependent DNA polymerase (29). We found that RNA-primed MPRCA blocked the synthesis of by-products derived from the primers and could amplify one molecule of circular DNA without using the submicroliter reaction volumes employed by Hutchison et al. (3). Because general ligation strategies circularize not only the desired DNA products but also undesired-products derived from self ligation (21), we also developed a ligation strategy to apply our amplification method in cell-free cloning of a ligated construct.Materials and methods Solutions, mixtures, plasticware, and DNA
To avoid contamination from the laboratory environment (24,30), solutions supplied by the manufacturers were used as much as possible in this study. UltraPURE distilled water (dDW) and 1 M Tris-HCl (pH 7.5) were purchased from Invitrogen (Carlsbad, CA, USA), and 0.5 M EDTA was from NipponGene (Tokyo, Japan). Similarly, the plasticware used had already been sterilized by the supplier: aerosol resistant filter tips were purchased from Molecular BioProducts (San Diego, CA, USA), and Biopur microcentrifuge tubes and PCR-grade 0.2-mL tubes were from Eppendorf (Hamburg, Germany). Purified pUC19 was purchased from Takara Bio (Otsu, Shiga, Japan); λ phage DNA was from TOYOBO (Osaka, Japan); and 1-kb DNA ladder marker was from New England BioLabs (Ipswich, MA, USA). Other solutions and reaction mixtures were prepared using a dedicated set of pipets (Finnpipette Focus; Thermo Fisher Scientific, Vantaa, Finland) in a dedicated clean bench after cleaning using RNase AWAY (Molecular Bioproducts, San Diego, CA, USA).