Whole Genome Amplification (MDA-WGA) and Rolling Circle Amplification (RCA)

Phi29 DNA polymerase and EquiPhi29 DNA polymerase are the main enzymes of choice for WGA and RCA. Thermo Scientific EquiPhi29 DNA polymerase is a proprietary Phi29 DNA polymerase mutant developed through in vitro protein evolution. [1] This enzyme is significantly superior over Phi29 in protein thermostability, reaction speed, product yield, and amplification bias. Moreover, it retains all the benefits of the wild-type enzyme, including high processivity (up to 70 kb), strong strand displacement activity, and 3′→5′ exonuclease (proofreading) activity. For this reason, exo-resistant primers are recommended.

Table 1. Comparison of Phi29 and EquiPhi29 DNA polymerases with supporting data.

In studies comparing other commercially available versions of Phi29 DNA polymerases, EquiPhi29 DNA polymerase demonstrated the lowest bias when amplifying targets with GC-rich content (Figure 2) and delivered the highest yield of a target sequence from whole genomic DNA (Figure 3) within 2 hours.

In recent years, the application of RCA in the amplification of small circular DNA has unlocked new possibilities for the utilization of strand-displacing polymerases, notably the Phi29 and Equiphi29 DNA polymerases. RCA can be used in a broad spectrum of applications such as DNA sequencing, cell-free DNA enrichment, and cell-free protein expression. RCA plays an important role in nucleic acid detection, analysis of pathogens, analysis of genetic mutations, food products, and environmental objects, and the development of mRNA vaccines.

RCA is a simple and highly efficient method to specifically amplify circular DNA targets for downstream sequencing analysis. Also, unspecific nucleic amplification by random hexamer priming of circular templates is used prior to NGS. This workflow is used in conventional research; for example, RCA enables fast Sanger sequencing of plasmid clones without the need for plasmid preparation(**). It is the essential amplification method in cancer diagnostics [5] and metagenomics.

In NGS-based on nanopore technology, debranching of the Phi29 generated molecules is necessary to enable molecule size control. Debranching is performed by T7 endonuclease I enzyme prior to sequencing [6].

RCA is employed for DNA enrichment, by distinguishing and amplifying circular DNA. This approach aids the amplification of the DNA of interest from a pool of genomic DNA. This method is often applied to amplify circular viral genomes using a specific primer. 

RCA-based DNA enrichment is also important for viral and microbial metagenomic studies. Entire viral and microbial populations can be newly identified starting with RCA, followed by sequencing and data analysis. 

Lastly, mitochondrial small circular genome is an ideal target for DNA enrichment. Therefore, RCA is utilized in mitochondria-based evolutionary or disease research [7].

Direct RCA is a technique used to amplify specific DNA sequences within bacterial cells without the need for DNA extraction. The first step of direct RCA is releasing DNA template from bacterial cells. To implement this step, bacterial colonies should be lysed in water with a short heating step that denatures the DNA template and facilitates the annealing of random primers. Random priming enables the synthesis of both strands of DNA, leading to the production of a double-stranded product. Amplified RCA products can be used for in vitro cloning, library construction, high-throughput cell-free protein expression, and other molecular biology applications [8].

Cell-free protein expression provides a simple and effective method of generating proteins, eliminating the challenges associated with cell culture, cell engineering, or cell transfection. Numerous limitations encountered in cell-based expression systems are overcome by using cell-free systems to express recombinant proteins. These limitations include issues such as protein toxicity, protein degradation, protein aggregation, misfolding, and uncontrolled post-translational modification.

A cell-free protein expression system offers the potential for expressing significantly larger quantities of proteins in a shorter period. This system can be effectively utilized for subsequent high-throughput structural and functional analyses. In vitro, protein expression provides benefits in terms of cost reduction, streamlined production, simplified purification, and easier scalability. In a cell-free protein expression system, the desired protein is synthesized by introducing a DNA or RNA molecule containing the gene sequence of interest into a transcription-translation-competent cellular extract. The gene is then transcribed and/or translated, with the option to couple both processes in a single reaction, allowing immediate translation of newly synthesized mRNA into protein. 

Cell-free protein systems require a large amount of DNA template, typically in microgram quantities. Moreover, the circular template is more suitable for this method, because of nucleases that are present in cell-free transcription-translation extracts that can degrade linear DNA sequences. To generate large quantities of high-quality DNA with less effort, time, and expense, isothermal amplification technology such as RCA can be used [9].
 

1. Povilaitis, T., Alzbutas, G., Sukackaite, R., Siurkus, J., & Skirgaila, R. (2016). In vitro evolution of phi29 DNA polymerase using isothermal compartmentalized self replication technique. Protein Engineering Design & Selection, 29(12), 617–628.
2. Zhang, L., Cui, X., Schmitt, K., Hubert, R. B., Navidi, W., & Arnheim, N. (1992). Whole genome amplification from a single cell: implications for genetic analysis. Proceedings of the National Academy of Sciences of the United States of America, 89(13), 5847–5851.
3. Dean, F. B., Hosono, S., Fang, L., Wu, X., Faruqi, A. F., Bray-Ward, P., Sun, Z., Zong, Q., Du, Y., Du, J., Driscoll, M., Song, W., Kingsmore, S. F., Egholm, M., & Lasken, R. S. (2002). Comprehensive human genome amplification using multiple displacement amplification. Proceedings of the National Academy of Sciences of the United States of America, 99(8), 5261–5266.
4. Blanco, L., Bernad, A., Lázaro, J. M., Martín, G., Garmendia, C., & Salas, M. (1989). Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. The Journal of biological chemistry, 264(15), 8935–8940.
5. Marano, J. M., Chuong, C., & Weger-Lucarelli, J. (2020). Rolling circle amplification: A high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones. Virology, 551, 58–63.
6. Ke, R., Mignardi, M., Hauling, T., & Nilsson, M. (2016). Fourth Generation of Next-Generation Sequencing Technologies: Promise and Consequences. Human mutation, 37(12), 1363–1367.
7. Jaberi, E., Tresse, E., Grønbæk, K., Weischenfeldt, J., & Issazadeh-Navikas, S. (2020). Identification of unique and shared mitochondrial DNA mutations in neurodegeneration and cancer by single-cell mitochondrial DNA structural variation sequencing (MitoSV-seq). EBioMedicine, 57, 102868. 
8. Dean, F. B., Nelson, J. R., Giesler, T. L., & Lasken, R. S. (2001). Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome research, 11(6), 1095–1099.
9. Hadi, T., Nozzi, N., Melby, J. O., Gao, W., Fuerst, D. E., & Kvam, E. (2020). Rolling circle amplification of synthetic DNA accelerates biocatalytic determination of enzyme activity relative to conventional methods. Scientific reports, 10(1), 10279.