Phi29-based DNA Rolling Circle Amplification

          Rolling Circle Amplification (RCA) is an isothermal enzymatic process, which amplifies circular or the extended linear genomic DNA by a Phi29 DNA polymerase-based rolling circle mechanism in the presence of the short DNA or RNA primers, as shown in Fig. 1. Phi29 DNA polymerase is a monomeric protein with two distinct functional domains, C-terminal DNA polymerase domain for DNA synthesis and a spatially separated N-terminal domain with a 3'-5' exonuclease activity for proofreading activity. Co-operation as well as delicate competition between two functional domains, ensure the accurate and efficient DNA synthesis at an optimal rate. Furthermore, this enzyme has the capacity to strongly bind to single stranded DNA of double stranded nucleic acid, which makes it favorable for multiple displacement amplification (MDA), through debranching double stranded DNA. RCA technology is better than PCR-based methods for DNA amplification because of the high processivity and proofreading activity of Phi29 polymerase. Specially, when genomic DNA is amplified, RCA generates larger fragments (more than 10 kb) with better coverage and less amplification bias, than PCR-based approaches. The RCA technology is so simple, powerful, and versatile that it has been extensively used for DNA cloning, sequencing, SNP and STR genotyping, and genomic DNA amplification, etc. Since the amplified DNA is a concatemer containing tens to thousands of tandem repeats, RCA technology has been employed to generate complex but fine DNA nanostructures, such as DNA origami, nanotubes, and nanoribbons, through manipulating the circular tailor-designed template. These DNA nanostructures have critical potentials in biomedical research, disease diagnostics and therapeutics.

 

          Through optimizing RCA reaction condition and engineering Phi29 enzyme, two classes of RCA DNA amplification technologies, RCA1.0 and RCA2.0 as described below, were developed for the various applications. SuperPhi DNA Amplification Kit, a type of RCA1.0 technology, was suitable for DNA cloning, direct DNA sequencing, and regular DNA amplification; while RCA2.0 technology would be specifically desired for circle DNA/RNA detection, single cell genomic amplification, cell-free DNA library construction etc.

Fig. 1 Schematic diagram of the RCA process. Random hexamer primers anneal to the circular single strand DNA at multiple sites. Phi29 DNA polymerase extends with highest fidelity. With the progress of reaction, Phi29 will extend on the synthesized DNA and amplify DNA thousand folds.

RCA 1.0 Technology:

 

          Our SuperPhi DNA Amplification Kit (RCA 1.0) was developed to rapidly amplify circular template DNA. The amplified DNA, as high molecular weight and double-stranded concatemers of the circular template, can be used for cloning and direct DNA sequencing without further purification. The DNA template can be low or high copy plasmid from bacterial colony, liquid culture, glycerol stock, DNA from BAC culture, M13 plaque, M13 Phage culture supernatant, fosmids or any lambda vectors, etc. Our highly pure SuperPhi enzyme in SuperPhi DNA Amplification Kit ensures that the amplified DNAs are from your template DNA, but less likely from the host or environmental DNAs, which were usually found in other Phi29-based DNA amplification kits. This amplification technology also eliminates the need for overnight cell culture and conventional plasmid or M13 preparations.

 

          Features of our SuperPhi DNA Amplification Kit (RCA1.0):

  • SuperPhi29 DNA polymerase for isothermal multiple displacement amplification (MDA);

  • Clean manufacturing process ensures components are free of detectable DNA contamination;

  • Proprietary enzymatic clean-up and UV step removes DNA contamination introduced before amplification;

  • Greater accuracy than existing other PCR based MDA methods;

  • Able to detect down to 1 picogram of circular DNA or genomic DNA.

 

          SuperPhi DNA Amplification (RCA) Kit with Random Primers (Cat#: Phi100) and its derived SuperPhi RCA with Specific Primers (Cat#: Phi100S), SuperPhi RCA Premix Kit with Random Primers (Cat#: PM100), SuperPhi RCA premix with Specific Primers (Cat#: PM100S) all are suitable for amplifying circular DNA from: 

  • Purified DNA or DNA ligation/assembly reactions;

  • Bacterial colonies on plate;

  • Liquid bacterial culture;

  • Glycerol stock.

 

          The amplified DNA by RCA1.0 technology is suitable for: 

  • Direct DNA restriction enzyme digestion without further purification;

  • Direct DNA sequencing (Sanger's method) without further purification;

  • Essential for Bacillus plasmid transformation.

Fig. 2. Comparison of purity and activity of our Powerful SuperPhi with Phi29 from competitor G.  The indicated amount of denatured pUC19 were mixed with random DNA primer (upper panel) and random RNA primer (low panel) in the presentence of SuperPhi and Phi29 from competitor G. After 4 hrs at 30oC, the reaction was stopped and loaded on agarose gel. The intensity of 15 kb amplified product using SuperPhi did response well to the input dosage of pUC19, while that in competitor G did not (in both upper and low panels).  Even on condition of no input pUC19, the reaction containing Phi29 from competitor G still produced amplified DNA when using RNA primer (in low panel). That indicated that competitor Phi29 enzyme was contaminated with foreign DNA.

RCA 2.0 Technology:

 

          RCA technology was widely accepted to amplify circle or genomic DNA. However, there were always around 20% of the amplified DNA product that can’t be mapped to any organism in the sequence database (Fig. 2), even with the most optimized reaction conditions containing RNA primer (Fig. 2). Particularly, when single cell genomic DNA or very low amount of the template DNA, such as less than 10 picogram, was recruited to amplify, this non-specific amplified DNA would represent up to 50% of total amplified DNA (Fig. 2). The presence of the non-specific amplified DNA could deteriorate the correct data interpretation, and severely interfere with the downstream application. It was presumed that the nonspecific RCA products were most likely derived from false priming at sites where primer dimers are formed. To avoid this non-specific DNA amplification due to the false priming in RCA reaction when using random primers, we thus applied protein engineering approach to modify the C-terminal domain of Phi29 polymerase. This resulting Phi29 polymerase (patent pending), called as FemtoPhi, prevented the extension from the short false primer dimer, and significantly decreased the non-specific DNA amplification in RCA reaction, and improved DNA amplification efficiency over wild type Phi29 enzyme (Fig. 3). Furthermore, the FemtoPhi DNA Amplification Kit (RCA2.0) had capacity to detect 10 plasmid DNA molecules (1 femtogram), which is almost 1000 fold sensitive over wild type Phi29, and was comparable to PCR or qPCR (Table 1). FemtoPhi DNA Amplification (RCA) Kit with Random Primers (Cat#: FP100), FemtoPhi RCA Premix Kit with Random Primers (Cat#: FM100), FemtoPhi RCA premix with Specific Primers (Cat#: FM100S) all are developed for various applications.

      

Fig.3. Comparison of Efficiency and Specificity of SuperPhi DNA Amplification Kit and FemtoPhi DNA Amplification Kit. The indicated amount of denatured pCT-eGFP (copy number) were mixed with the corresponsive SuperPhi (upper panel) and FemtoPhi (lower panel), respectively. After the reaction, the products were directly digested with EcoR1 and then loaded on agarose gel. The Red Arrowhead indicated the expected products, while the Green Arrow pointed to the non-specific amplification products. The non-specific primers and left primers (White Arrowhead) were found in FemtoPhi DNA amplification reactions (lower panel), but could not in SuperPhi DNA Amplification reactions (upper panel). It was assumed that the non-specific primers were used to amplify DNA in SuperPhi Kit, resulting in the smear bands (upper panel). The lowest copy number of plasmid can be detected in FemtoPhi Kit was 10 copies (~0.1 femtogram), while 10^5 copies (~1 picogram) in SuperPhi Kit. The 10^6 copies of plasmid are equivalent to 10 picogram.

 

Table 1. Brief Comparison of PCR and our RCA.

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