Consistent Temperature. Consistent Results.
The need for fast, field-deployable molecular assays has never been greater. Recombinase Polymerase Amplification (RPA) is a highly sensitive isothermal amplification method that enables rapid, onsite pathogen testing, including for infectious diseases. However, enzyme and protein lot-to-lot variability has historically limited its reliability — until now.
Key Features and Benefits
- Amplify consistently with individually QC'd enzymes and proteins, improving performance and reproducibility
- Overcome LAMP limitations with lower amplification temperatures, simple primer design, and shorter amplification times
- Go mobile with glycerol-free, lyophilization-friendly formulations, enabling robust field-based testing
Applications
- Infectious disease detection
- Field-based and point-of-care testing
- Rapid multiplex panels
- Sample-to-answer and integrated systems
Figure 1. RPA molecular diagram ... MORE
Recombinase Polymerase Amplification
Figure 1. Molecular representation of Recombinase Polymerase Amplification (RPA). T4 UvsX DNA Recombinase (green ovals) and T4 UvsY Protein (purple hexagons) bind to amplification primers, forming a complex that is complementary to sequences in double stranded DNA. T4 Gene 32 Protein (pink dots) acts as a single-stranded binding protein and stabilizes the unwound DNA strand, allowing Bsu DNA Polymerase - Large Fragment (red ovals) to initiate strand displacing amplification. The repetition of the cycle leads to exponential amplification. Schematic recreated from James, et al, Diagnostics 2020, 10, 399.
Proteins Available
| Protein | Details |
| T4 UvsX DNA Recombinase (UvsX) | Quality controlled for enzyme activity (forming D-loop recombination structures for initiation of amplification) and freeze/thaw stable |
| T4 UvsY Protein (UvsY) | Accessory protein to UvsX with robust single stranded binding |
| T4 Gene 32 Protein (gp32) | Stabilizes ssDNA to increase amplification efficiency and primer specificity |
| Bsu DNA Polymerase, Large Fragment (Bsu) | Displays strong strand displacement and enables isothermal amplification at lower temperatures than Bst polymerase |
Key Performance Data
Rigorous manufacturing and quality control
Reliable RPA results start with consistent enzyme performance – something the field has lacked until now.
Individual activity-based QC assays have been developed and implemented for each RPA enzyme to precisely control concentration and activity, ensuring consistent production and performance.
Rigorous manufacturing and quality control
Inhibitor tolerance enables direct amplification
For assays developed for field or point-of-care testing, direct amplification without nucleic acid purification saves precious time and reduces costs. Watchmaker RPA enzymes reliably amplify across diverse sample types and tolerate inhibitors commonly found in extracted nucleic acids.
Figure 2. Amplify efficiently in the presence of inhibitors ... MORE
Inhibitor tolerance enables direct amplification
Figure 2. Amplify efficiently in the presence of inhibitors. A variety of common inhibitors were added to RPA reactions amplifying a 168 bp target. In all cases, RPA demonstrates strong tolerance to inhibitors routinely encountered in nucleic acid extraction methods or biological samples.
Fast amplification over a wide dynamic range
Due to the variable nature of field-collected samples, the ability to detect targets across a broad dynamic range is essential. Watchmaker RPA enzymes deliver rapid and consistent amplification across a wide concentration range, with sensitivity ≤ 100 copies and reactions reaching peak fluorescence in under 10 minutes.
Fast amplification over a wide dynamic range
Stable performance through multiple freeze-thaw cycles
To combat the common issue of freeze-thaw instability, Watchmaker's RPA enzymes are supplied in specially-formulated glycerol-free storage buffers that not only allow lyophilization — but also survive multiple freeze/thaw cycles without loss of activity.
Figure 4. Maintain activity across freeze-thaw events ... MORE
Stable performance through multiple freeze-thaw cycles
Figure 4. Maintain activity across freeze-thaw events. Enzymes were freeze-thaw cycled 30 times (-80°C to room temperature). Activity was measured using Watchmaker enzyme-specific QC assays.
BROCHURES
Recombinase Polymerase Amplification Brochure
Custom Genomic Solutions Isothermal Amplification Pathogen Detection Enzymes MDx RNase H T4 Gene 32 Protein Bsu DNA Polymerase, Large Fragment Recombinase Polymerase Amplification (RPA) T4 UvsX DNA Recombinase T4 UvsY ProteinThis brochure presents the Watchmaker Recombinase Polymerase Amplification (RPA) enzymes including T4 UvsX DNA Recombinase, T4 UvsY Protein, T4 Gene 32 Protein, and Bsu DNA Polymerase, Large Fragment.
Custom Genomic Solutions Brochure
Custom Genomic Solutions Isothermal Amplification Pathogen Detection PCR/qPCR Rolling Circle Amplification RT-PCR/RT-qPCR Enzymes MDx Equinox Library Amplification Equinox Uracil-Tolerant Library Amplification phi29 DNAP RNase Inhibitor RNase H StellarScript RT StellarScript HT RT StellarScript HT+ RT StellarTaq DNA Polymerase T4 DNA Ligase T4 DNAP T4 Polynucleotide Kinase T4 Gene 32 Protein Taq DNAP Bsu DNA Polymerase, Large Fragment Recombinase Polymerase Amplification (RPA) T4 UvsX DNA Recombinase T4 UvsY ProteinWhether you need small-scale modifications or a fully custom solution, Watchmaker’s flexible model ensures you get exactly what you need — with speed, simplicity, and scientific precision.
POSTERS
Optimization of Recombinase Polymerase Amplification (RPA) highlights the importance of enzyme consistency for reproducible results
Isothermal Amplification DNA Enzymes MDx T4 Gene 32 Protein Bsu DNA Polymerase, Large Fragment Recombinase Polymerase Amplification (RPA) T4 UvsX DNA Recombinase T4 UvsY ProteinThis poster presents data showing how Watchmaker individual enzyme QC for RPA enzymes leads to reliable, consistent amplification. Effects of damaged proteins in RPA reactions are measured, as well as the effect of multiple freeze-thaw cycles. As presented at ADLM 2025.
Evaluation of Recombinase Polymerase Amplification (RPA) for suitability in molecular diagnostic applications
Isothermal Amplification Enzymes MDx Recombinase Polymerase Amplification (RPA)This poster presents data demonstrating the speed, sensitivity, inhibitor tolerance, and multiplexing capability of Recombinase Polymerase Amplification (RPA). Data is also shown for reactions in which StellarScript HT+ Reverse Transcriptase was added, enabling one-step isothermal amplification of viral RNA targets, including in multiplex reactions.
PROTOCOLS
T4 Gene 32 Protein Technical Guide
Enzymes MDx T4 Gene 32 Protein Recombinase Polymerase Amplification (RPA)Protocol for T4 Gene 32 Protein (gp32) is a single-stranded DNA-binding protein from T4 bacteriophage that stabilizes ssDNA, enhancing yields in molecular biology assays like PCR and RT-PCR.
Bsu DNA Polymerase Technical Guide
Custom Genomic Solutions Isothermal Amplification Pathogen Detection Enzymes MDx Bsu DNA Polymerase, Large Fragment Recombinase Polymerase Amplification (RPA)Technical guide for Bsu DNA Polymerase, Large Fragment (Bsu). Bsu is chiefly used with other enzymes in Recombinase Polymerase Amplification (RPA), as it exhibits strong strand displacement activity and enables isothermal amplification at lower temperatures than Bst polymerase.
T4 UvsX DNA Recombinase - Glycerol-Free Technical Guide
Custom Genomic Solutions Isothermal Amplification Pathogen Detection Enzymes MDx Recombinase Polymerase Amplification (RPA) T4 UvsX DNA RecombinaseTechnical guide for T4 UvsX DNA Recombinase (UvsX). UvsX is chiefly used with other enzymes in Recombinase Polymerase Amplification (RPA), where it facilitates priming of the amplification site.
T4 UvsY Protein - Glycerol-Free Technical Guide
Custom Genomic Solutions Isothermal Amplification Pathogen Detection Enzymes MDx Recombinase Polymerase Amplification (RPA) T4 UvsY ProteinTechnical guide for T4 UvsY Protein (UvsY). UvsY is chiefly used with other enzymes in Recombinase Polymerase Amplification (RPA), where it functions by stabilizing and enhancing the activity of UvsX, the primary recombinase enzyme.
| QUALITY CONTROL | UvsX, UvsY, Bsu, and gp32 |
|---|---|
| Purity (SDS-PAGE) | ≥ 95% (gp32 ≥ 98%) |
| dsDNA exonuclease | < 1% released |
| ssDNA exonuclease | < 1% released |
| DNA contamination Assay (E. coli, mammalian, library)* | < 10 copies |
| Phosphatase Contamination Assay* | < 1% released |
| Endonuclease Contamination Assay* | Not detectable |
| Activity Assay | Passed |
* As assessed using 5 μg of UvsX; 2 μg of UvsY; 25 mg of gp32; or 50 U of Bsu.
| SHIPPING AND HANDLING | UvsX, UvsY, Bsu, and gp32 |
|---|---|
| Shipping | Dry Ice |
| Storage | -80°C ± 10°C |
What is T4 UvsX DNA Recombinase (UvsX)?
UvsX is a recombinase that is analogous to the bacterial RecA protein. UvsX serves an important role in homologous recombination, and in the case of isothermal amplification methods, the enzyme facilitates the priming of the amplification site when utilized in the presence of a DNA template and primer oligos. This enzyme, in combination with T4 UvsY Protein (UvsY) and T4 Gene 32 Protein (gp32) can be used to form recombination complexes, which then allow Bsu DNA Polymerase to initiate polymerization via the recombinase polymerase amplification (RPA) method.
What is T4 UvsY Protein (UsvY)?
UvsY is an accessory enzyme derived from bacteriophage T4 that plays a critical role in recombinase polymerase amplification (RPA), an isothermal DNA amplification technique. It functions by stabilizing and enhancing the activity of UvsX, the primary recombinase enzyme, facilitating the formation of nucleoprotein filaments on single-stranded DNA.
Are T4 UvsX DNA Recombinase and T4 UvsY Protein available in glycerol-free format?
Yes, UvsX and UvsY are available in glycerol-free format only, which is suitable for downstream lyophilization.
What are the recommended T4 UvsX DNA Recombinase and T4 UvsY Protein storage conditions?
Store at -70°C to -90°C.
Are Watchmaker’s T4 UvsX DNA Recombinase and T4 UvsY Protein freeze-thaw stable?
Yes, UvsX and UvsY are freeze-thaw stable. Contact support@watchmakergenomics.com for further information.
What are the relevant applications for T4 UvsX DNA Recombinase and T4 UvsY Protein?
Recombinase Polymerase Amplification (RPA).
What components are required for Recombinase Polymerase Amplification (RPA)?
To achieve optimal RPA performance and reliability, use T4 UvsX DNA Recombinase (7K0124), T4 UvsY Protein (7K0126), T4 Gene 32 Protein (7K0127) and Bsu DNA Polymerase, Large Fragment (7K0128). Please refer to the UvsX or UvsY Technical Guides for other components required for RPA which should be sourced separately.
How do I design primers for RPA?
The following characteristics are of critical importance when designing RPA primers:
- The optimal length for RPA primers is 30–35 nt. Primers less than 30 nt are not recommended.
- Primer sequences should have between 30–70% GC content and have no single or dinucleotide base repeats.
- Primer sequences should also be devoid of complementary sequences which promote secondary structure hairpin loops and prevent self-dimerization or primer-primer interactions.
- Amplicon sequences should have between 35–60% GC content, with an optimal length of 150–450 bp.
- If possible, the primer should end with a G or C on the 3' terminus.
How are T4 UvsX DNA Recombinase and T4 UvsY Protein inactivated?
Incubation at 75°C for 20 minutes will inactivate UvsX or UvsY.
Where can I go for additional help or technical support?
If you need more detailed guidance or have questions not addressed here, please reach out to our support team at support@watchmakergenomics.com or submit a request via our Technical Support form. Please include details such as product name, lot number, version of protocol you’re using, and a brief description of your question or issue so we can assist you promptly.
Do you offer custom formats?
Yes! Watchmaker offers customer fills, packaging, concentrations, and labeling - including private label - designed to meet your unique needs. We offer flexible terms to serve organizations of any size, and our right-sized processes enable rapid turnaround time on customization. Please contact sales@watchmakergenomics.com to learn more about our capabilities.
Are you ISO 13485-certified?
Yes! Our Quality Management System has achieved ISO 13485:2016 Certification. Our certificate has been awarded for the design, development, manufacture, contract manufacture, and support of high performing reagents for genomics applications in medical research. Download the certificate here.
| Description | Part no. | Pack size | |
|---|---|---|---|
| T4 UvsX DNA Recombinase - Glycerol-free (5 mg/mL) | 7K0124-25UL 7K0124-500UL | 125 µg 2,500 µg | Request a quote |
| T4 UvsY Protein - Glycerol-free (2 mg/mL) | 7K0126-25UL 7K0126-500UL | 50 µg 1,000 µg | Request a quote |
| Bsu DNA Polymerase, Large Fragment - Glycerol-free (50 U/μL) | 7K0128-25UL 7K0128-500UL | 1.25 kU 25 kU | Request a quote |
| T4 Gene 32 Protein - Glycerol-free (10 mg/mL) | 7K0127-25UL 7K0127-500UL | 250 µg 5,000 µg | Request a quote |
| RPA Enzyme Sample Pack - Glycerol-free | 7K0125-25UL | 25 µL of each enzyme | Request a quote |
Please contact sales@watchmakergenomics.comto inquire about custom kit configurations.
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