Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification

Abstract

We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques. Using random primers and 29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods and traditional DNA isolation methods. Reaction products can be used directly for DNA sequencing after phosphatase treatment to inactivate unincorporated nucleotides. Amplified products can also be used for in vitro cloning, library construction, and other molecular biology applications.A fundamental requirement for molecular biology is the isolation and amplification of specific DNA sequences. Target sequences are typically inserted into circular vectors, propagated in a biological host, and isolated by physical methods (Sambrook et al. 1989). However, such methods are laborious, costly, and not amenable to high-density formats. PCR is also used to amplify defined sequences, but can introduce sequence errors and is limited to amplification of short DNA segments (Innis et al. 1990).In nature, the replication of circular DNA molecules such as plasmids or viruses frequently occurs via a rolling circle mechanism (Kornberg and Baker 1992). As a laboratory method, linear rolling circle amplification (RCA) (Fire and Xu 1995;Liu et al. 1996;Lizardi et al. 1998) is the prolonged extension of an oligonucleotide primer annealed to a circular template DNA. A continuous sequence of tandem copies of the circle is synthesized. RCA has the advantage of not requiring a thermal cycling instrument. Two primers are used to perform exponential (or hyperbranched) RCA, one for each strand (Lizardi et al. 1998). A cascade of strand displacement reactions results in an exponential amplification.Previously, RCA had been used to amplify small DNA circles approximately 100 nt in length. However, the rate for plasmid-sized targets is only about 20 copies per hour, limiting the usefulness with plasmids or other circles larger than 0.2 kb. We describe here a technique called multiply-primed rolling circle amplification (multiply-primed RCA) that uses the unique properties of 29 DNA polymerase and random primers to achieve a 10,000-fold amplification. This robust process allows amplification of circular DNA directly from cells or plaques, generating high-quality template for use in DNA sequencing, probe generation, or cloning. The method is simple and is optimally performed at 30°C, making it suitable for a variety of applications. This will make it attractive for high-throughput processes and 384-well formats. RESULTS Increased Yield in RCA Using Random Hexamer PrimersIn multiply-primed RCA, the use of multiple primers annealed to a circular template DNA generates multiple replication forks (Fig. 1). RCA proceeds by displacing the nontemplate strand. In this way, product strands are "rolled off" of the template as tandem copies of the circle. Random priming allows synthesis of both strands, resulting in double-stranded product. ...

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