PrimeTime^® LNA^® (locked nucleic acid) qPCR Probes

LNA bases can be incorporated into dual-labeled probes (DLPs) [1–4]. Because LNA bases significantly increase T_m, LNA PrimeTime LNA qPCR Probes can be designed with shorter lengths than standard DLPs. 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 LNA DLP can be designed to have a ΔT_m of >15°C, which greatly increases accuracy of allele determination in real-time PCR or other methods that use differential hybridization to distinguish polymorphisms.

PrimeTime LNA qPCR Probes can be ordered either at a guaranteed yield of 0.5 nmol or at defined synthesis scales to suit your research needs:

PrimeTime Mini LNA qPCR Probes

• Ideal for analyzing a small sample set or performing a few reactions to optimize probe designs
• Available with FAM, HEX™ or YAK^® fluorescent dyes, and Iowa Black^® FQ quencher
• Guaranteed normalized yield of 0.5 nmol
• Shipped in 4–6 business days

PrimeTime LNA 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, insertion of an LNA base into a DNA oligo can increase the T_m by 3–6°C.
• The relative binding affinity (T_m) of LNA bases are LNA:LNA > LNA:DNA > DNA:DNA. Therefore, it is important to examine the probe sequence for self-dimer and hairpin formation and minimize designs that allow LNA:LNA pairing. Use the OligoAnalyzer^® Tool to check secondary structure predictions.
• Amplicon sizes up to 150 bp are recommended for standard qPCR, and shorter amplicons of 80–100 bp are typically recommended for digital PCR (dPCR) or applications with shortened fragments (such as FFPE or small circulating DNA).
• We recommend using up to 6 LNA bases in an LNA dual-labeled probe:
  • 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 LNA bases on the first or last bases of the probe sequence.
  • Additional LNA bases can be incorporated to adjust T_m, as needed.
  • Placement of the LNA bases is sequence dependent, and may require optimization to achieve and optimal ∆T_m between match and mismatch. IDT recommends placing no more than 4 LNA bases sequentially.

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