gene_expression

Affinity Plus qPCR Probes

Increase SNP target specificity and use shorter sequences with these high Tm probes

Use Affinity Plus qPCR Probes for SNP genotyping, transcript variant identification, and more sensitive target detection in challenging samples (FFPE tissue, biofluids). The Affinity Plus bases used in these qPCR probes include up to 6 locked nucleic acid monomers (see the technology page, Locked nucleic acids, for more information). When incorporated into a probe, they impart heightened structural stability, leading to increased hybridization melt temperature (Tm).

  • Improve mismatch discrimination compared to traditional qPCR probes
  • Benefit from this affordable approach to increasing assay hybridization specificity
  • Make flexible Tm adjustments more easily than possible with MGB probes

Ordering

Prices listed include probe sequence (10–25 standard bases with up to 6 Affinity Plus bases), reporter, quencher, and HPLC purification. Affinity Plus qPCR Probes are typically shipped within 4–6 business days. See the Product Details tab for design tips.

Mini Affinity Plus qPCR Probes

Mini Affinity Plus qPCR Probes are ideal for screening small sample sets or performing just a few reactions when optimizing probe designs.

5' Reporter Dye(s)Quencher(s)Delivery Amount
0.5 nmol
FAMIowa Black FQ *€ 70,00 EUR
HEXIowa Black FQ *€ 70,00 EUR
YAKIowa Black FQ *€ 70,00 EUR

Affinity Plus qPCR Probes

Affinity Plus qPCR Probes are offered with a wider selection of dyes and quenchers than Affinity Plus Mini qPCR Probes, and are best-suited for large-scale or high-throughput applications.

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Advantages of Affinity Plus qPCR Probes

Locked nucleic acid bases, such as Affinity Plus monomers, can be incorporated into effective qPCR probes [1–4]. Because these 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 probes offer an improved ability to distinguish mutations or single nucleotide polymorphisms (SNPs) [1]. An Affinity Plus qPCR Probe can be designed to have 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

Design considerations

  • Depending on sequence context, each Affinity Plus base insertion into a DNA oligo will increase the Tm by 3–6°C. Multiple additions can result in an Affinity Plus qPCR Probe with a ΔTm of >15°C.
  • The relative binding affinity (Tm) of Affinity Plus 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 bases in an Affinity Plus qPCR Probe.
    • Affinity Plus 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 bases on the first or last bases of the probe sequence.
    • Additional Affinity Plus bases can be incorporated to adjust Tm as needed. We recommend placing no more than 4 Affinity Plus bases sequentially.
    • Placement of the Affinity Plus bases is sequence 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 DNA samples).

For additional assistance with designing Affinity Plus qPCR Probes, email applicationsupport@idtdna.com. Design fees may apply.

References

  1. Davialieva K, Kiprijanovska S, Plaseska-Karanfilska D. (2013) Fast, reliable and low cost user-developed protocol for detection, quantification and genotyping of hepatitis C virus. J Virol Methods, 196:104–112.
  2. Owczarzy R, You Y, et al. (2011) Stability and mismatch of locked nucleic acid–DNA duplexes. Biochemistry, 50(43):9352–9367.
  3. You Y, Moreira BG, et al. (2006) Design of LNA probes that improve mismatch discrimination. Nucl Acid Res 34(8):e60, doi:10.1093/nar/gkl175.
  4. Johnson MP, Haupt LM, Griffiths LR. (2004) Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR. Nucleic Acids Res, 32(6):e55.




Frequently asked questions