Pushing the Limits of Sensitivity
Highly sensitive & specific padlock probes
superRCA® mutation kits are one of the first commercially available flow cytometry-based assay that uses padlock probes that enable ultra-sensitive Measurable Residual Disease (MRD) evaluation in hemato-oncology research. This powerful approach helps ensure:- Ultra-high sensitivity across a broad range of target genes (e.g., LoD NPM1-1:100000)
- Wide DNA input range (5-2,650 ng)
- Recognition of SNPs in challenging GC-rich sequences (e.g., ASXL1)
- High sample efficiency (660 ng are sufficient for an LoD of 1x10-5)
How it works
superRCA® technology uses four interdependent mechanisms to improve genotyping sensitivity:| 1. Probe hybridization
(binding to the matched target sequence) |
|
Expert designed probes |
| 2. Ligase-mediated allele distinction
(only perfect match base pair can be ligated into replicable circles) |
|
Control Mechanism 1 |
| 3. Circle confirmation
(only a successfully ligated probe can contribute to a superRCA® reaction) |
|
Control Mechanism 2 |
| 4. Majority voting mechanism
(the concatemer-based padlock probing employs hundreds to thousands of padlock probes to sense the same target molecule) |
|
Further suppression of errors |
Explore a step-by-step tour of the superRCA® technology
Comparison of dPCR and superRCA® assay fidelity
The superior target specificity of superRCA® technology is achieved by a very high stringency of the probe binding to the target sequence. PCR-based detection relies on the TaqMan probe hybridization only. The specificity is DNA sequence-dependent and can vary. superRCA® assays have a very high stringency of probe binding with four independent mechanisms that ensure high fidelity (see section above).Target Specificity: qPCR, dPCR vs superRCA®
| Target Specificity | qPCR, dPCR | superRCA® |
| Probes | TaqMan | Padlock |
| Mechanism that ensures specificity | Probe Hybridization | 1. Probe Hybridization 2. Ligase-mediated allele distinction 3. Circle Confirmation 4. Majority voting mechanism |
| Stringency of probe binding | Low (DNA sequence dependent) | Very High (4 mechanisms for high fidelity) |
PCR-based DNA amplification is exponential but every error that is made by the DNA Polymerase is amplified exponentially. dPCR relies on Taq DNA-polymerase that is known for its high error rate. superRCA® assays use High Fidelity (HF) polymerase that has a 1000 times lower error rate. Furthermore, RCA is a linear amplification method, and therefore errors are not amplified.
Method Accuracy: qPCR, dPCR vs superRCA®
| Method Accuracy | qPCR, dPCR | superRCA® |
| Polymerase error rate | Taq (10-4) | HF (10-7) |
| PCR cycles (exponential amplification) | Up to 40 x | 10 x |
| Method-based errors | High (exponential amplification of errors) | Very Low |
dPCR-based assays are limited by the number of compartments. superRCA® assays leverage the statistical power and speed of flow cytometry. With 30 times more data points compared to dPCR, superRCA® technology achieves very low CV values and gives you higher confidence in your data.
Variations: qPCR, dPCR vs superRCA®
| Variations | qPCR, dPCR | superRCA® |
| Data points | 22,000 | 400,000 - 1,300,00 |
| CV | High | Very Low |
| Technical Replicates | 2-3 | Not Required |
