gene_expression

PrimeTime® LNA® qPCR Probes

Shorter probes with high Tm and increased specificity compared to traditional 5′ nuclease probes

LNA probes are commonly used for genotyping and custom applications such as transcript variant detection or differential detection of microbial species. LNA is a modified nucleic acid monomer. When incorporated into a probe, LNA imparts heightened structural stability to the target sequence leading to increased hybridization temperature.

  • Choose from a wide variety of reporter and quencher combinations
  • Available in small scale for discovery studies
  • Improve mismatch discrimination compared to traditional probes

Ordering

See Product details for design considerations or email applicationsupport@idtdna.com for design assistance.

Prices listed include probe sequence (10–25 standard bases with up to 6 LNA bases), reporter, quencher, and HPLC purification. LNA probes are typically shipped within 4–6 business days.

PrimeTime Mini LNA qPCR Probes

PrimeTime Mini LNA qPCR Probes are ideal for screening a small sample set or performing a few reactions to optimize probe designs.

5' Reporter Dye(s)Quencher(s)Delivery Amount
0.5 nmol
FAMIowa Black FQ *$135.00 USD
HEXIowa Black FQ *$135.00 USD
YAKIowa Black FQ *$135.00 USD

PrimeTime LNA qPCR Probes

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

LNA bases can be incorporated into dual-labeled probes (DLPs) [1–4]. Because LNA bases significantly increase Tm, 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 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.

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 Tm by 3–6°C.
  • The relative binding affinity (Tm) 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 Tm, as needed.
    • Placement of the LNA bases is sequence dependent, and may require optimization to achieve and optimal ∆Tm between match and mismatch. IDT recommends placing no more than 4 LNA bases sequentially.

For additional assistance with designing PrimeTime LNA 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