What is it?
BaseBlockers™ are short oligonucleotides modified with several pentabases to ensure tight and specific binding to a sequence that should be inhibited from working as a template. They are furthermore modified to ensure that they do not work as potential primers. Hence, by adding BaseBlockers™, one can avoid false positive and false negative signals.
Principle of BaseBlockersTM
BaseBlockers™ work by binding strongly and specifically to one allele (for example to a wild type allele), while allowing amplification of the other allele. A BaseBlocker™ binds to a fully complementary sequence, thereby inhibiting the primer from binding and the template from being amplified.
Key features
- Very efficient in removing false positive signals
- Little to no impact on mutation signals
- Can remove cross signals
- Significantly increases the robustness of an assay
- Increases the detection range of your target
BaseBlocker™ assay principle
SNV detection assay using a green fluorescent HydrolEasy™ probe (light green), allele-specific forward primers (black) and a BaseBlocker™ (red). The allele-specific forward primers include the sequence variation in the 3´-end (green) and an additional sequence variation close to the 3´-end (purple).
The BaseBlocker™ is complementary to the wild type sequence (yellow) and specifically blocks amplification of the wild-type allele. From Riva et al. 2017.
Benefits of BaseBlockersTM
Adding BaseBlockers™ to your assay not only reduces the false positive signals from the presence of WT templates. BaseBlockers™ also reduce the demand for stringency allowing the assay to be conducted on different instruments, in different laboratories and by different operators. Furthermore, it increases the detection range of your target of interest. That is why we call BaseBlockers™ a universal PCR specifier.
BaseBlockers™ are made customized to your assay and can be purchased in 50, 200 or 1,000 nmol synthesis scales. Just design an oligo spanning the site you need to block with the same affinity as your primer and ask for a BaseBlocker™.
The figure below, shows how a well-designed BaseBlocker™ can inhibit the false positive signal generated by a WT template, with no influence on the mutation specific assay.
We offer solutions for microsatellite instability analysis. We cover both Bethesda and Hamelins panel.