BNA bases have a modification to the ribose backbone that locks the base in the C3′-endo conformation, which favors RNA A-type helix duplex geometry. This modification significantly increases Tm and is also relatively nuclease resistant.
BNAs can be incorporated into dual-labeled probes for use in 5′ nuclease assays. Because BNA bases significantly increase Tm, PrimeTime BNA Probes can be designed with shorter lengths than standard dual-labeled probes. Shorter probes are more effectively quenched and have a higher signal-to-noise ratio; therefore, they are more sensitive.
More importantly, these probes offer an improved ability to distinguish mutations or single nucleotide polymorphisms (SNPs). A DNA dual-labeled probe typically has a ΔTm of ~5°C between a perfect-match and mismatched hybridization. In contrast, a BNA dual-labeled probe can have a ΔTm of >15°C, greatly increasing accuracy of allele determination in real-time PCR or other methods that use differential hybridization to distinguish polymorphisms.
Tm calculations can be made using the OligoAnalyzer® Tool. The LNA notation (+T, +A, +G, +C) to estimates Tm or mismatch discrimination potential of BNA bases. BNA probe design points to consider include:
- Insertion of a BNA base into a DNA oligo can increase the Tm by 3–6°C, depending on sequence context. However, there are some sequence-specific designs involving G-T mismatches where BNA bases may impair specificity.
- We recommend including up to 6 BNA bases in a BNA dual-labeled probe:
- BNA bases should be placed at the SNP site and adjacent bases.
- The SNP should be positioned in the center of the probe, if possible.
- Additional BNA bases can be incorporated toward the 3′-end of the probe to adjust Tm, as needed.
- The relative binding affinity (Tm) of BNA bases are BNA:BNA > BNA:DNA > DNA:DNA. Therefore, it is important to examine the probe sequence for self-dimer and hairpin formation and minimize designs that allow BNA:BNA pairing.
For additional assistance with design of PrimeTime BNA Probes, email firstname.lastname@example.org (design fees may apply).