Advantages of Affinity Plus qPCR Probes containing LNA modifications
Locked nucleic acid (LNA) bases, such as Affinity Plus monomers, can be incorporated into effective qPCR probes [1–4]. Because LNA bases significantly increase Tm, Affinity Plus qPCR Probes can be designed with shorter lengths than standard
qPCR probes. Shorter probes have better quenching and a higher signal-to-noise ratio and are, therefore, more sensitive. More importantly, these LNA probes offer an improved ability to distinguish mutations or single nucleotide polymorphisms (SNPs) .
An Affinity Plus qPCR Probe can be designed with several LNA monomers resulting in a ΔTm of >15°C, which greatly increases
accuracy of allele determination in real-time PCR or other methods that use differential hybridization to distinguish polymorphisms.
Affinity Plus qPCR Probes can be ordered either at a guaranteed yield of 0.5 nmol or at defined synthesis scales to suit your research needs.
Mini Affinity Plus qPCR Probes
- Ideal for analyzing small sample sets or performing a few reactions when optimizing probe designs
- Available with FAM, HEX™, or Yakima Yellow® (YAK) fluorescent dyes, and Iowa Black FQ Quencher
- Guaranteed normalized yield of 0.5 nmol
- Shipped in 4–6 business days
Affinity Plus qPCR Probes
- Available with FAM, Cy® 3, Cy 5, TEX, TYE™, YAK, and HEX dyes
- High synthesis scales (250 nmol and 1 µmol) for large-scale and high throughput requirements
- Shipped in 4–6 business days
- Depending on sequence context, each LNA base insertion into a DNA oligo will increase the Tm by 3–6°C. Multiple LNA additions can result in an Affinity Plus qPCR Probe with a ΔTm of >15°C.
- The relative binding affinity (Tm) of Affinity Plus LNA bases are Affinity Plus:Affinity Plus > Affinity Plus:DNA > DNA:DNA. Therefore, it is important to examine the probe sequence for self-dimer and
hairpin formation, and minimize designs that allow Affinity Plus:Affinity Plus pairing. Use the OligoAnalyzer Tool to check secondary structure predictions.
- We recommend using up to 6 Affinity Plus LNA bases in an Affinity Plus qPCR Probe.
- Affinity Plus LNA bases should be placed at the SNP site and on each of the adjacent bases. The SNP should be positioned in the center of the probe, if possible. Avoid placing Affinity Plus LNA bases on the first or last bases of the probe sequence.
- Additional Affinity Plus LNA bases can be incorporated to adjust Tm as needed. We recommend placing no more than 4 Affinity Plus LNA bases sequentially.
- Placement of the Affinity PlusLNA bases is
dependent, and may require optimization to achieve optimal ∆Tm between match and mismatch.
- Amplicon sizes up to 150 bp are recommended for standard qPCR. Shorter amplicons of 80–100 bp are typically recommended for digital PCR (dPCR) or for applications with samples containing shortened fragments (such as FFPE or small circulating
For additional assistance with designing Affinity Plus qPCR Probes, email firstname.lastname@example.org. Design fees may apply.