RCA and MDA-WGA | Thermo Fisher Scientific

This graphic depicts nucleic acid strand displacement, the technique used in whole genome amplification and rolling circle amplification to amplify nucleic acids.

Whole genome amplification (WGA) is a molecular biology technique used to amplify the entire genome even from very small amounts of DNA—even a single cell—to obtain large quantities of product. This method is based on multiple displacement amplification. When the starting material is circular DNA, rolling circle amplification (RCA) is utilized. Both MDA-WGA and RCA reactions are performed under isothermal conditions and use strand-displacing polymerases, such as phi29 DNA polymerase.

Rolling circle amplification (RCA)

The process of RCA involves the use of a circular DNA template, usually in the form of a plasmid or a circularized oligonucleotide. This template is amplified by a DNA polymerase enzyme, such as phi29 DNA polymerase, that binds to the template to synthesize new DNA strands. As the DNA polymerase moves around the circular template, it continuously synthesizes new copies of DNA, leading to exponential amplification. This process can be repeated multiple times to generate large amounts of DNA from a small starting sample.

Schematic illustration of strand displacement activity and subsequent DNA amplification in rolling circle amplification

Figure 1. Rolling circle amplification (RCA) is an isothermal amplification technique where long single-stranded DNA is produced from a circular DNA template using a strand-displacing DNA polymerase, such as phi29 DNA Polymerase. RCA generates a concatemer that contains numerous tandem repeats that are complementary to the circular template.

Multiple displacement amplification–whole genome amplification (MDA–WGA)

MDA-WGA uses a highly processive DNA polymerase, such as phi29 DNA polymerase, to amplify DNA at a constant temperature. The process starts with the addition of random hexamer primers to the DNA sample, which hybridize to the template DNA and initiate DNA synthesis.

Schematic illustration of strand displacement activity and subsequent DNA amplification in multiple displacement amplification – whole genome amplification

Figure 2: Multiple displacement amplification–whole genome amplification (MDA–WGA). Schematic representation of whole genome amplification that is based on multiple displacement amplification. The diagram shows strand-displacing activity by DNA polymerase, such as phi29 polymerase.

Enzymes for MDA-WGA and RCA

phi29 DNA polymerase and EquiPhi29 DNA polymerase are the enzymes of choice for MDA-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 superior to wild-type phi29 DNA polymerase 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.

Table 1. Comparison of EquiPhi29 and phi29 DNA Polymerases

	EquiPhi29 DNA Polymerase	phi29 DNA Polymerase
Processivity/strand displacement	High	High
Optimal amplification temperature	42°C	30°C
Reaction time	2 h	4—16 h
Proofreading activity	3‘→5‘	3'→5‘
Fidelity	High	High
Sensitivity	1 fg—1 ng	1 pg—1 ng
Yield	Very high	High
GC sequence bias	Very low	Low
Format	Stand-alone enzyme DNA amplification kit	Stand-alone enzyme

EquiPhi29 DNA Polymerase

Thermo Scientific EquiPhi29™ DNA Polymerase is a proprietary phi29 DNA Polymerase mutant that possesses strong strand displacement activity that allows for fast, sensitive, and efficient isothermal amplification such as rolling circle amplification (RCA) and multiple displacement amplification–whole genome amplification (MDA-WGA). Compared to wide type phi29 DNA Polymerase, EquiPhi29 DNA Polymerase has increased thermostability, reaction speed, product yield, and lower amplification bias. EquiPhi29 DNA Polymerase is available in stand-alone and kit formats.

Highlights:

Fast—Amplifies target in less than 2 hours
Sensitive—Achieves sensitivity from 1 fg of DNA
High yield—Yields up to 17 µg amplified DNA
Variety of samples—Different types of sample input material (e.g., purified DNA, liquid media culture, agar plate colonies)
Variety of applications—Amplified product can be used with various downstream applications (DNA sequencing [e.g., Sanger sequencing, next-gen sequencing], digestion with restriction enzymes, cell-free DNA enrichment, and cell-free protein expression)

EquiPhi29 DNA Amplification Kit

The EquiPhi29 DNA Polymerase is also available as a part of the Thermo Scientific EquiPhi29 DNA Amplification Kit. This kit includes all of the components required for efficient isothermal DNA amplification, including rolling circle amplification (RCA) and multiple displacement amplification-whole genome amplification (MDA-WGA).

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RCA data using EquiPhi29 DNA Polymerase

Thermostability and product yield

EquiPhi29 DNA Polymerase helps ensure higher thermostability, reaction speed, and product yield compared to wild type phi29 DNA polymerase (Figures 3 and 4).

graph comparing equiphi29 and phi29 DNA Polymerases' yields at 25–45°C using pUC19 plasmid. EquiPhi29 shows higher yield across temperatures.

Figure 3.EquiPhi29 DNA Polymerase provides greater product yield at higher working temperatures compared to wild type phi29 DNA polymerase. DNA amplification was performed with the EquiPhi29 DNA Amplification kit and Thermo Scientific phi29 DNA Polymerase at different temperatures (25–45°C) for 2 hours using 0.1 ng of pUC19 plasmid according to the product protocol. Amplified DNA was quantified using the Invitrogen Quant-iT PicoGreen dsDNA Assay Kit. In all cases, the data represent the mean of RCA yield obtained from n=3 replicates with standard deviation.

Graph showing superior yield and reaction speed of EquiPhi29 DNA Polymerase compared to Thermo Scientific™ phi29 and other enzymes.

Figure 4. EquiPhi29 DNA Polymerase provides high plasmid DNA yields with faster reaction times than other commercially available phi29 DNA polymerases. Amplification of 0.1 ng pUC19 plasmid DNA was performed for 1–4 hours using EquiPhi29 DNA Amplification Kit at different temperatures (30, 42, and 45°C), Thermo Scientific phi29 DNA Polymerase at 30°C and other supplier kits at recommended conditions. Amplified DNA was quantified using the Invitrogen Quant-iT PicoGreen dsDNA Assay Kit. In all cases, the data represents the mean of RCA yield obtained from n=3 replicates with standard deviation.

High sensitivity

The high sensitivity of EquiPhi29 DNA Polymerase enables amplification from a limited amount of starting material of the target DNA. EquiPhi29 DNA Polymerase shows higher or similar sensitivity and product yield compared to other commercially available kits. EquiPhi29 DNA Polymerase achieves sensitivity from 1 fg of plasmid DNA (Figure 5).

Graph comparing RCA yields of EquiPhi29 kit versus other vendors with 1fg, 1pg, 1ng pUC19 DNA, showing higher sensitivity and yield of EquiPhi29 from minimal DNA amounts.

Figure 5. High sensitivity and product yield from low amounts of DNA. pUC19 plasmid DNA, in amounts of 1 fg, 1 pg and 1 ng were used as an input in RCA reactions with EquiPhi29 DNA Amplification Kit according to the product protocol. RCA kits from other vendors were used according to the manufacturers’ protocols. RCA products were quantified using the Invitrogen Quant-iT PicoGreen dsDNA Assay Kit. In all cases, the data represent the mean of RCA yield obtained from n=3 replicates with standard deviation.

RCA use in cell-free protein expression

Combination of Gibson assembly or gene self-circularization with RCA using EquiPhi29 DNA Amplification kit enables DNA construction and cell-free protein expression in 1 day (Figure 6).

Graphs comparing traditional plasmid preparation technique vs plasmid preparation using EquiPhi29 DNA Amplification kit for Sanger sequencing and cell-free protein expression

Figure 6. Comparison of traditional plasmid preparation technique vs plasmid preparation using EquiPhi29 DNA Amplification kit for Sanger sequencing and cell-free protein expression.

EquiPhi29 DNA Polymerase ensures fast and efficient circular DNA amplification in RCA-enabled cell-free protein expression application (Figure 7).

Graph displaying comparable protein synthesis efficiency between RCA products from single colony, Gibson assembly, self-circularization, and non-amplified plasmid

Figure 7. Products of direct RCA from a single colony, Gibson assembly, or self-circularization reaction yield as efficient cell-free protein synthesis as non-amplified plasmid. 1 µL of RCA reactions or 500 ng of purified plasmid was used for protein synthesis with RTS 100 E. coli HY Kit (Biotech Rabbit) according to the manufacturer’s recommendations. Protein expression was monitored by measuring GFP fluorescence every 5 minutes. All RCA products are expressed at the same level as non-amplified purified plasmid.

RCA data using Lyo-ready Bst DNA Polymerase

Figure 8. Lyo-ready Bst DNA Polymerase produces more amplified product compared to other Bst polymerases. Fluorescence signal intensity was determined in the RCA reaction from 1 ng, 5 ng, and 10 ng inputs of circular ssDNA template, 1:1 primer and ssDNA ratio was used. The RCA was performed at 65°C for 1 h. The fluorescence of the hybridized beacon was determined using Applied Biosystems QuantStudio 7 Flex Real-Time PCR System, where it was normalized to no template control. Normalized fluorescence is positively correlated to the amount of RCA product. In all cases, the data represent the mean of RCA yield obtained from n=4 replicates with standard deviation.

MDA-WGA data using EquiPhi29 DNA Polymerase

Low GC content bias

Thermo Scientific EquiPhi29 DNA polymerase demonstrates the lowest bias when amplifying targets with GC-rich content (Figure 9).

Graph comparing GC bias by EquiPhi29, phi29, and other DNA polymerases. EquiPhi29 shows uniform coverage across varying GC contents, outperforming others in minimizing GC bias.

Figure 9. EquiPhi29 DNA Polymerase ensures low GC bias when amplifying different bacterial genomes. A mixture of bacterial genomes with low-GC (S. aureus, 33% GC), moderate-GC (E. coli, 51% GC), and high-GC (P. aeruginosa, 68% GC) content was amplified using EquiPhi29 and phi29 DNA polymerases as well as a DNA polymerase from another supplier. For each genome, the GC content of the reference genome, in 100 bp windows indicated in gray, was plotted versus the coverage normalized to the unamplified genome mix, indicated in green. In the absence of sequencing bias, all windows should be equally distributed close to the normalized coverage of 1, indicated in light blue. The normalized coverage obtained after amplification using different polymerases is shown. EquiPhi29 DNA Polymerase amplifies DNA with the lowest GC bias across all GC contents when compared to other DNA polymerases.

High genomic DNA yield

EquiPhi29 DNA polymerase delivers a high yield of a target sequence from human genomic DNA (Figure 10) within 2 hours.

Figure 10. EquiPhi29 DNA Polymerase provides high genomic DNA yields with faster reaction times than wild type phi29 DNA Polymerase. DNA amplification was performed with the EquiPhi29 DNA Amplification Kit at different temperatures (30, 42, and 45 °C) and Thermo Scientific phi29 DNA Polymerase at 30 °C for 1–4 hours using 0.1 ng of human genomic DNA according to the product protocol. Amplified DNA was quantified using the Invitrogen Quant-iT PicoGreen dsDNA Assay Kit. In all cases, the data represents the mean of MDA-WGA yield obtained from n=3 replicates with standard deviation.

phi29 DNA Polymerase

Thermo Scientific phi29 DNA Polymerase is a highly processive polymerase featuring strong strand displacement activity, which allows for efficient isothermal DNA amplification such as RCA and MDA-WGA.

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Developing a WGA or RCA assay?

Thermo Fisher Scientific stand-alone enzymes include the polymerase, buffer, and other additional components to enable maximal flexibility in reaction setup and the feasibility of developing assays for MDA-WGA or RCA applications. They can be customized in the glycerol-free lyo-ready format. Click below to request a quote for custom products like Lyo-ready EquiPhi29 DNA Polymerase for MDA-WGA and RCA.

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phi29 DNA polymerase

EquiPhi29 DNA polymerase

References:

Usage	Reference
Padlock probe- and rolling circle amplification-based RNA in situ hybridization assay for detecting HPV E6/E7 mRNA expression.	Rao, X., Zheng, L., Wei, K., Li, M., Jiang, M., Qiu, J., Zhou, Y., Ke, R., & Lin, C. (2023). Novel In Situ Hybridization Assay for Chromogenic Single-Molecule Detection of Human Papillomavirus E6/E7 mRNA. Microbiology Spectrum, 11(2).
Isothermal amplification of Plasmodium falciparum genomic DNA using selective whole genome amplification (sWGA).	Choubey, D., Deshmukh, B., Rao, A., Kanyal, A., Hati, A. K., Roy, S., & Karmodiya, K. (2023). Genomic analysis of Indian isolates of Plasmodium falciparum: Implications for drug resistance and virulence factors. International Journal for Parasitology-Drugs and Drug Resistance, 22, 52–60.
Enrichment of T. pallidum genomic DNA using selective whole genome amplification (sWGA).	Thurlow, C. M., Joseph, S. J., Ganova-Raeva, L., Katz, S. S., Pereira, L., Chen, C., Debra, A., Vilfort, K., Workowski, K. A., Cohen, S., Reno, H., Sun, Y., Burroughs, M., Sheth, M., Chi, K., Danavall, D., Philip, S., Cao, W., Kersh, E. N., & Pillay, A. (2022). Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing. mSphere, 7(3).
Enrichment of N. meningitidis DNA using improved selective whole genome amplification (sWGA).	Itsko, M., Topaz, N., Ousmane, S., Popoola, M., Ouédraogo, R., Gamougam, K., Sadji, A. Y., Abdul-Karim, A., Lascols, C., & Wang, X. (2022). Enhancing meningococcal genomic surveillance in the meningitis belt using High-Resolution Culture-Free Whole-Genome sequencing. The Journal of Infectious Diseases, 226(4), 729–737.
Padlock probe-based rolling circle amplification of Mycobacterium tuberculosis DNA followed by real-time OM detection.	Minero, G. a. S., Bagnasco, M., Fock, J., Tian, B., Garbarino, F., & Hansen, M. F. (2020). Automated on-chip analysis of tuberculosis drug-resistance mutation with integrated DNA ligation and amplification. Analytical and Bioanalytical Chemistry, 412(12), 2705–2710.

Usage	Reference
Rolling circle amplification of African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) DNA followed by Illumina Sequencing	Aimone, C. D., Lavington, E., Hoyer, J. S., Deppong, D. O., Mickelson-Young, L., Jacobson, A. L., Kennedy, G. G., Carbone, I., Hanley-Bowdoin, L., & Duffy, S. (2021). Population diversity of cassava mosaic begomoviruses increases over the course of serial vegetative propagation. Journal of General Virology, 102(7).
Padlock probe-based rolling circle amplification of Arabidopsis genes followed by imaging with the confocal microscope.	Nobori, T., Oliva, M., Lister, R., & Ecker, J. R. (2023). Multiplexed single-cell 3D spatial gene expression analysis in plant tissue using PHYTOMap. Nature Plants, 9(7), 1026–1033.
Rolling circle amplification of grapevine red blotch virus (GRBV) DNA followed by conventional PCR and Sanger sequencing.	Thompson, J. R. (2022). Analysis of the genome of grapevine red blotch virus and related grabloviruses indicates diversification prior to the arrival of Vitis vinifera in North America. Journal of General Virology, 103(10).

Usage	Reference
Rolling circle amplification of mRNA from breast cancer and liver cancer tissue using V-probe based vsmCISH method.	Jiang, M., Wei, K., Li, M., Lin, C., & Ke, R. (2023). Single molecule RNA in situ detection in clinical FFPE tissue sections by vsmCISH. RNA, 29(6), 836–846.
Isothermal amplification of cell-free DNA from cancer patients using blunt end ligation-mediated whole genome amplification (BL-WGA) followed by mutational analysis.	Tomeva, E., Switzeny, O. J., Heitzinger, C., Hippe, B., & Haslberger, A. G. (2022). Comprehensive Approach to Distinguish Patients with Solid Tumors from Healthy Controls by Combining Androgen Receptor Mutation p.H875Y with Cell-Free DNA Methylation and Circulating miRNAs. Cancers, 14(2), 462.

Research area	Usage	Reference
Method description for cell-free protein expression	Multiply-primed rolling circle amplification followed by cell-free protein expression.	Zibulski, D. L., Schlichting, N., & Kabisch, J. (2022). HyperXpress: Rapid single vessel DNA assembly and protein production in microliterscale. Frontiers in Bioengineering and Biotechnology, 10.
microRNA detection	Hairpin probe–assisted Isothermal circular strand displacement amplification of miRNA.	Bellassai, N., D’Agata, R., & Spoto, G. (2022). Isothermal circular strand displacement–based assay for microRNA detection in liquid biopsy. Analytical and Bioanalytical Chemistry, 414(22), 6431–6440.

[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.

Pathogen analysis

Usage	Reference
Padlock probe- and rolling circle amplification-based RNA in situ hybridization assay for detecting HPV E6/E7 mRNA expression.	Rao, X., Zheng, L., Wei, K., Li, M., Jiang, M., Qiu, J., Zhou, Y., Ke, R., & Lin, C. (2023). Novel In Situ Hybridization Assay for Chromogenic Single-Molecule Detection of Human Papillomavirus E6/E7 mRNA. Microbiology Spectrum, 11(2).
Isothermal amplification of Plasmodium falciparum genomic DNA using selective whole genome amplification (sWGA).	Choubey, D., Deshmukh, B., Rao, A., Kanyal, A., Hati, A. K., Roy, S., & Karmodiya, K. (2023). Genomic analysis of Indian isolates of Plasmodium falciparum: Implications for drug resistance and virulence factors. International Journal for Parasitology-Drugs and Drug Resistance, 22, 52–60.
Enrichment of T. pallidum genomic DNA using selective whole genome amplification (sWGA).	Thurlow, C. M., Joseph, S. J., Ganova-Raeva, L., Katz, S. S., Pereira, L., Chen, C., Debra, A., Vilfort, K., Workowski, K. A., Cohen, S., Reno, H., Sun, Y., Burroughs, M., Sheth, M., Chi, K., Danavall, D., Philip, S., Cao, W., Kersh, E. N., & Pillay, A. (2022). Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing. mSphere, 7(3).
Enrichment of N. meningitidis DNA using improved selective whole genome amplification (sWGA).	Itsko, M., Topaz, N., Ousmane, S., Popoola, M., Ouédraogo, R., Gamougam, K., Sadji, A. Y., Abdul-Karim, A., Lascols, C., & Wang, X. (2022). Enhancing meningococcal genomic surveillance in the meningitis belt using High-Resolution Culture-Free Whole-Genome sequencing. The Journal of Infectious Diseases, 226(4), 729–737.
Padlock probe-based rolling circle amplification of Mycobacterium tuberculosis DNA followed by real-time OM detection.	Minero, G. a. S., Bagnasco, M., Fock, J., Tian, B., Garbarino, F., & Hansen, M. F. (2020). Automated on-chip analysis of tuberculosis drug-resistance mutation with integrated DNA ligation and amplification. Analytical and Bioanalytical Chemistry, 412(12), 2705–2710.

Plant research

Usage	Reference
Rolling circle amplification of African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) DNA followed by Illumina Sequencing	Aimone, C. D., Lavington, E., Hoyer, J. S., Deppong, D. O., Mickelson-Young, L., Jacobson, A. L., Kennedy, G. G., Carbone, I., Hanley-Bowdoin, L., & Duffy, S. (2021). Population diversity of cassava mosaic begomoviruses increases over the course of serial vegetative propagation. Journal of General Virology, 102(7).
Padlock probe-based rolling circle amplification of Arabidopsis genes followed by imaging with the confocal microscope.	Nobori, T., Oliva, M., Lister, R., & Ecker, J. R. (2023). Multiplexed single-cell 3D spatial gene expression analysis in plant tissue using PHYTOMap. Nature Plants, 9(7), 1026–1033.
Rolling circle amplification of grapevine red blotch virus (GRBV) DNA followed by conventional PCR and Sanger sequencing.	Thompson, J. R. (2022). Analysis of the genome of grapevine red blotch virus and related grabloviruses indicates diversification prior to the arrival of Vitis vinifera in North America. Journal of General Virology, 103(10).

Cancer research

Usage	Reference
Rolling circle amplification of mRNA from breast cancer and liver cancer tissue using V-probe based vsmCISH method.	Jiang, M., Wei, K., Li, M., Lin, C., & Ke, R. (2023). Single molecule RNA in situ detection in clinical FFPE tissue sections by vsmCISH. RNA, 29(6), 836–846.
Isothermal amplification of cell-free DNA from cancer patients using blunt end ligation-mediated whole genome amplification (BL-WGA) followed by mutational analysis.	Tomeva, E., Switzeny, O. J., Heitzinger, C., Hippe, B., & Haslberger, A. G. (2022). Comprehensive Approach to Distinguish Patients with Solid Tumors from Healthy Controls by Combining Androgen Receptor Mutation p.H875Y with Cell-Free DNA Methylation and Circulating miRNAs. Cancers, 14(2), 462.

Additional areas

Research area	Usage	Reference
Method description for cell-free protein expression	Multiply-primed rolling circle amplification followed by cell-free protein expression.	Zibulski, D. L., Schlichting, N., & Kabisch, J. (2022). HyperXpress: Rapid single vessel DNA assembly and protein production in microliterscale. Frontiers in Bioengineering and Biotechnology, 10.
microRNA detection	Hairpin probe–assisted Isothermal circular strand displacement amplification of miRNA.	Bellassai, N., D’Agata, R., & Spoto, G. (2022). Isothermal circular strand displacement–based assay for microRNA detection in liquid biopsy. Analytical and Bioanalytical Chemistry, 414(22), 6431–6440.

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