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Recombinase Polymerase Amplification (RPA) |
Recombinase polymerase amplification (RPA) is a sensitive, low-temperature, isothermal DNA amplification technique that does not require thermal cycling. RPA is initiated by two primers and carried out by a mixture of recombinase enzymes, a single-stranded binding protein, and a strand-displacing DNA polymerase. RPA is a versatile technique with applications spanning from pathogen diagnostics to next-generation sequencing. It is performed at a constant temperature of 39–42°C, making it highly suitable for detecting various pathogens in field conditions. RPA can also be used to detect RNA targets by incorporating reverse transcriptase into the reaction. The reverse transcriptase synthesizes a cDNA template from RNA before the recombinase complex binds the primers to initiate the RPA reaction. Amplified DNA can be detected using various detection methods such as end-point detection, real-time analysis, lateral flow assays, and CRISPR-Cas systems.
Figure 1. Recombinase polymerase amplification (RPA). The RPA reaction starts when a recombinase complex made of UvsX and UvsY proteins binds to the primers in the presence of ATP. This complex seeks a homologous sequence on DNA and promotes strand opening and invasion by the primer. The single-strand DNA binding protein (Gene 32 Protein) stabilizes the resulting D-loop structure, allowing the DNA polymerase to extend the primer while displacing the DNA strand.
Invitrogen Lyo-ready RPA Kit
The Invitrogen Lyo-ready RPA Kit includes all required components in a glycerol-free format for isothermal recombinase polymerase amplification (RPA), enabling high reaction sensitivity, specificity, and tolerance to inhibitors. The Lyo-ready RPA Kit contains separate reagents that provide maximum flexibility to optimize the RPA reaction and work with various types of pathogens (RNA/DNA), including S. aureus, P. aeruginosa, influenza, measles, and other pathogens. Components for (RT-)RPA reactions are also available in a stand-alone format.
Product highlights:
- Sensitive—Achieves sensitivity as low as one copy of target samples
- Specific—Detects and amplifies a single target in a mixture of RNA/DNA
- Fast—Amplifies targets in as little as 20 minutes
- Robust—Amplifies even from inhibitor-containing RNA/DNA samples
- Flexible—Provides the ability to optimize the RPA or RT-RPA reaction
The high sensitivity of the Lyo-ready RPA Kit enables amplification from a limited amount of starting material or from samples containing low concentrations of the target DNA/RNA. The Lyo-ready RPA Kit demonstrates higher sensitivity than other commercially available RPA Kits.
Figure 2. High sensitivity and reliable amplification of 238 bp target with as low as 5 copies of P. aeruginosa genomic DNA. 238 bp target was amplified from 0.1 pg–1 ng of P. aeruginosa genomic DNA using Invitrogen Lyo-ready RPA Kit or other isothermal amplification kits according to manufacturer’s recommendations. Amplified products were analyzed on 2% E-Gel EX Agarose Gel. The molecular weight marker is E-Gel 1 Kb Plus Express DNA Ladder.
Figure 3. High sensitivity and reliable amplification of 386 bp target with as low as 5 copies of M. pneumoniae genomic DNA. 386 bp target was amplified from 0.01–100 pg of M. pneumoniae genomic DNA using Invitrogen Lyo-ready RPA Kit or other isothermal amplification kits according to manufacturer’s recommendations. Amplified products were analyzed on 2% E-Gel EX Agarose Gel. The molecular weight marker is E-Gel 1 Kb Plus Express DNA Ladder.
Figure 4. High sensitivity and reliable amplification of 320 bp target down to 0.01 ng of human genomic DNA. 320 bp target was amplified from 0.01–100 ng of human genomic DNA using Invitrogen Lyo-ready RPA Kit or other isothermal amplification kits according to manufacturer’s recommendations. Amplified products were analyzed on 2% E-Gel EX Agarose Gel. The molecular weight marker is E-Gel 1 Kb Plus Express DNA Ladder.
Figure 5. High sensitivity and reliable amplification of 161 bp target down to one copy of Adenovirus 41 DNA. 161 bp target was amplified from 1–1,000 copies of Adenovirus 41 DNA using Invitrogen Lyo-ready RPA Kit or other isothermal amplification kits according to manufacturer’s recommendations. Amplified products were analyzed on 2% E-Gel EX Agarose Gel. The molecular weight marker is E-Gel 1 Kb Plus Express DNA Ladder.
Figure 6. High sensitivity and reliable amplification of 206 bp target down to one copy of SARS-CoV-2 RNA. 206 bp target was amplified from 1–1,000 copies of SARS-CoV-2 RNA using Invitrogen Lyo-ready RPA Kit together with Invitrogen SuperScript IV Reverse Transcriptase, Invitrogen RNaseOUT Recombinant Ribonuclease Inhibitor, and Thermo Scientific RNase H, or other isothermal amplification kits according to manufacturer’s recommendations. Amplified products were analyzed on 2% E-Gel EX Agarose Gel. The molecular weight marker is E-Gel 1 Kb Plus Express DNA Ladder.
Figure 7. High sensitivity and reliable amplification of 253 bp target down to one copy of Zika virus RNA. 253 bp target was amplified from 1–1,000 copies of Zika virus RNA using Invitrogen Lyo-ready RPA Kit together with Invitrogen SuperScript IV Reverse Transcriptase, Invitrogen RNaseOUT Recombinant Ribonuclease Inhibitor and Thermo Scientific RNase H or other isothermal amplification kits according to manufacturer’s recommendations. Amplified products were analysed on 2% E-Gel EX Agarose Gel. The molecular weight marker is E-Gel 1 Kb Plus Express DNA Ladder.
The Lyo-ready RPA Kit demonstrates high specificity, enabling it to detect and amplify a single target in complex mixtures of genetic material. It helps ensure reliable results and minimizes the risk of non-specific products.
Figure 8. High target specificity from the pool of different DNA templates. Five different DNA templates were combined to a final concentration of 1,000 copies/reaction of Adenovirus 41 DNA, 1 ng/reaction of S. aureus genomic DNA, 1 ng/reaction of P. aeruginosa genomic DNA, 0.1 ng/reaction of M. pneumoniae genomic DNA, and 10 ng of human genomic DNA. Using target-specific primers and the Lyo-ready RPA Kit, each target was amplified specifically from the template that was surrounded with different unrelated DNA molecules. Amplified products (161 bp target from Adenovirus 41 DNA, 230 bp target from S. aureus genomic DNA, 238 bp target from P. aeruginosa genomic DNA, 305 bp target from human genomic DNA and 386 bp target from M. pneumoniae genomic DNA) were analyzed on 2% E-Gel EX Agarose Gel. The molecular weight marker is E-Gel 1 Kb Plus Express DNA Ladder.
Figure 9. High target specificity from the pool of different RNA templates. Five different RNA templates were combined to a final concentration of 1,000 copies/reaction of SARS-CoV-2 RNA, 1,000 copies/reaction of Zika virus RNA, 1,000 copies/reaction of Influenza H1N1 RNA, 100X diluted purified Chikungunya virus RNA, and 1,000 copies/reaction of Measles virus RNA. Using target-specific primers and the Lyo-ready RPA Kit—together with Invitrogen SuperScript IV Reverse Transcriptase, Invitrogen RNaseOUT Recombinant Ribonuclease Inhibitor, and Thermo Scientific RNase H—each target was amplified specifically from the template that was surrounded with different unrelated RNA molecules. Amplified products (192 bp target from SARS-CoV-2 RNA, 253 bp target from Zika virus RNA, 280 bp target from Influenza H1N1 RNA, 313 bp from Chikungunya virus RNA, and 452 bp from Measles RNA) were analyzed on 2% E-Gel EX Agarose Gel. The molecular weight marker is E-Gel 1 Kb Plus Express DNA Ladder.
RNA and DNA samples may have suboptimal purity even after a thorough sample purification process. The Lyo-ready RPA Kit demonstrates exceptional tolerance to common inhibitors originating from reagents used in RNA/DNA extraction steps or transferred from biological samples. (see Table 1).
Table 1. Common (RT-)RPA inhibitors and their sources.
| Inhibitor | Source |
|---|---|
| Ethanol | Sample preparation |
| Heparin, hemin, urea | Blood, urine |
| Humic acid, xylan | Soil, plants |
UTM (universal transport media) | Clinical specimens transported in UTM |
Figure 10. High performance in the presence of inhibitors. 305 bp target was amplified from 10 ng of human genomic DNA using Invitrogen Lyo-ready RPA Kit. Corresponding inhibitors were added to the prepared reaction mixes. Amplified products were analyzed on 2% E-Gel EX Agarose Gel. The molecular weight marker is E-Gel 1 Kb Plus Express DNA Ladder.
With high specificity and robustness, the Lyo-ready RPA Kit can successfully multiplex targets without the need for significant optimization steps.
Figure 11. High specificity of RPA and RT-RPA multiplex reactions.Three targets from different DNA templates were amplified in the same RPA reaction (238 bp target from 1 ng of P. aeruginosa genomic DNA, 305 bp target from 1 ng of human genomic DNA, and 376 bp target from 1 ng of S. aureus genomic DNA). Four targets from different RNA templates were amplified in the same RT-RPA reaction (192 bp target from 1,000 copies of SARS-CoV-2 RNA, 253 bp target from 1,000 copies of Zika virus RNA, 313 bp target from 100X diluted purified Chikungunya virus RNA, and 452 bp from 1,000 copies of Measles virus RNA). Multiplex reactions were performed using Invitrogen Lyo-ready RPA Kit with 0.1 µM final concentration of each primer, and incubated for 25 min at 42°C. In the case of RT-RPA reactions, Invitrogen SuperScript IV Reverse Transcriptase, Invitrogen RNaseOUT Recombinant Ribonuclease Inhibitor, and Thermo Scientific RNase H were additionally used, and reactions were incubated for 25 min at 45°C. Amplified products were analyzed using 2% E-Gel EX Agarose Gel. The molecular weight marker is E-Gel 1 Kb Plus Express DNA Ladder.
Lyo-ready RPA Kit is highly versatile and compatible with various detection systems, including real-time detection. Real-time detection allows for immediate monitoring and analysis of RPA products.
Figure 12. Real-time RT-RPA detection. 192 bp target was amplified from 10–1,000 copies of SARS-CoV-2 RNA with three repeats. Real-time RT-RPA reaction was performed using the Lyo-ready RPA Kit together with SuperScript IV Reverse Transcriptase, RNaseOUT Recombinant Ribonuclease Inhibitor, RNase H, and 5 µM of SYTO 9 green fluorescent nucleic acid stain for real-time detection.
Stand-alone RPA proteins and reagents
Invitrogen Lyo-ready T4 UvsX Protein
The Invitrogen Lyo-ready T4 UvsX Protein is a recombinase that plays an important role in the repair of double-stranded DNA breaks and the restart of replication forks. It forms helical assemblies on single-stranded DNA and promotes joint molecule formation between homologous DNA molecules.
In combination with its accessory proteins—Lyo-ready T4 UvsY and Lyo-ready T4 Gene 32 Protein—Lyo-ready T4 UvsX Protein can be used in isothermal recombinase polymerase amplification reaction (RPA) to form recombination complexes that initiate amplification by Lyo-ready Bst DNA Polymerase.
The Invitrogen Lyo-ready T4 UvsX Protein is a glycerol-free enzyme, enabling assay lyophilization and use in microfluidics-based technologies.
Invitrogen Lyo-ready T4 UvsY Protein
The Invitrogen Lyo-ready T4 UvsY Protein is a recombination mediator protein that enhances the ability of T4 UvsX recombinase to catalyze strand exchange and homologous recombination.
Lyo-ready T4 UvsX Protein, in combination with its accessory protein Lyo-ready T4 UvsY and Lyo-ready T4 Gene 32 Protein, can be used in an isothermal recombinase polymerase amplification reaction (RPA) to form recombination complexes that initiate amplification by Lyo-ready Bst DNA Polymerase.
The Invitrogen Lyo-ready T4 UvsY Protein is a glycerol-free enzyme, enabling assay lyophilization and use in microfluidics-based technologies.
Invitrogen Lyo-ready T4 Gene 32 Protein
The Invitrogen Lyo-ready T4 Gene 32 Protein, also known as a single-stranded DNA binding protein, is useful in many commonly used molecular biology techniques, such as PCR, cloning, and isothermal amplification like recombinase polymerase amplification reaction (RPA), where it stabilizes the D-loop structure and allows the DNA polymerase to extend the primer, displacing the DNA strand.
The Invitrogen Lyo-ready T4 Gene 32 Protein is a glycerol-free enzyme, enabling assay lyophilization and use in microfluidics-based technologies.
Invitrogen Lyo-ready Bst DNA Polymerase
The Invitrogen Lyo-ready Bst DNA Polymerase is a proprietary mutant of Bst DNA Polymerase, Large Fragment, demonstrating enhanced speed, strong strand displacement activity, and tolerance to inhibitors. In RPA, the Lyo-ready Bst DNA Polymerase amplifies the recombination complex formed by the Lyo-ready T4 UvsX Protein in combination with its accessory proteins Lyo-ready T4 UvsY and Lyo-ready T4 Gene 32 Protein.
Lyo-ready Bst DNA Polymerase is a glycerol-free enzyme, enabling assay lyophilization and use in microfluidics-based technologies.
Invitrogen Lyo-ready 2X RPA Reaction Buffer
The Invitrogen Lyo-ready 2X RPA Reaction Buffer enables a reliable and robust recombinase polymerase amplification (RPA) and is included in the Lyo-ready RPA Kit (Cat. No. A72127). In cases where additional quantities is required, 2X RPA Reaction Buffer is also made available in a stand-alone format.
Invitrogen Lyo-ready T4 UvsX Protein
The Invitrogen Lyo-ready T4 UvsX Protein is a recombinase that plays an important role in the repair of double-stranded DNA breaks and the restart of replication forks. It forms helical assemblies on single-stranded DNA and promotes joint molecule formation between homologous DNA molecules.
In combination with its accessory proteins—Lyo-ready T4 UvsY and Lyo-ready T4 Gene 32 Protein—Lyo-ready T4 UvsX Protein can be used in isothermal recombinase polymerase amplification reaction (RPA) to form recombination complexes that initiate amplification by Lyo-ready Bst DNA Polymerase.
The Invitrogen Lyo-ready T4 UvsX Protein is a glycerol-free enzyme, enabling assay lyophilization and use in microfluidics-based technologies.
Invitrogen Lyo-ready T4 UvsY Protein
The Invitrogen Lyo-ready T4 UvsY Protein is a recombination mediator protein that enhances the ability of T4 UvsX recombinase to catalyze strand exchange and homologous recombination.
Lyo-ready T4 UvsX Protein, in combination with its accessory protein Lyo-ready T4 UvsY and Lyo-ready T4 Gene 32 Protein, can be used in an isothermal recombinase polymerase amplification reaction (RPA) to form recombination complexes that initiate amplification by Lyo-ready Bst DNA Polymerase.
The Invitrogen Lyo-ready T4 UvsY Protein is a glycerol-free enzyme, enabling assay lyophilization and use in microfluidics-based technologies.
Invitrogen Lyo-ready T4 Gene 32 Protein
The Invitrogen Lyo-ready T4 Gene 32 Protein, also known as a single-stranded DNA binding protein, is useful in many commonly used molecular biology techniques, such as PCR, cloning, and isothermal amplification like recombinase polymerase amplification reaction (RPA), where it stabilizes the D-loop structure and allows the DNA polymerase to extend the primer, displacing the DNA strand.
The Invitrogen Lyo-ready T4 Gene 32 Protein is a glycerol-free enzyme, enabling assay lyophilization and use in microfluidics-based technologies.
Invitrogen Lyo-ready Bst DNA Polymerase
The Invitrogen Lyo-ready Bst DNA Polymerase is a proprietary mutant of Bst DNA Polymerase, Large Fragment, demonstrating enhanced speed, strong strand displacement activity, and tolerance to inhibitors. In RPA, the Lyo-ready Bst DNA Polymerase amplifies the recombination complex formed by the Lyo-ready T4 UvsX Protein in combination with its accessory proteins Lyo-ready T4 UvsY and Lyo-ready T4 Gene 32 Protein.
Lyo-ready Bst DNA Polymerase is a glycerol-free enzyme, enabling assay lyophilization and use in microfluidics-based technologies.
Invitrogen Lyo-ready 2X RPA Reaction Buffer
The Invitrogen Lyo-ready 2X RPA Reaction Buffer enables a reliable and robust recombinase polymerase amplification (RPA) and is included in the Lyo-ready RPA Kit (Cat. No. A72127). In cases where additional quantities is required, 2X RPA Reaction Buffer is also made available in a stand-alone format.
Developing an RPA assay?
Thermo Fisher Scientific stand-alone protein and reagents include the polymerase, buffer, and other additional components to enable maximal flexibility in reaction setup and the feasibility of developing assays for RPA applications. They can be customized in the glycerol-free, lyo-ready format. Click below to request a quote for custom RPA reactions.
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