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Oligonukleotid szintézis, szintetikus biológia

GENOTIPIZÁLÁS

LNA PrimeTime® Probes

 

LNA PrimeTime® Probes are dual-labeled DNA probes designed for use in 5’ nuclease assays. LNA PrimeTime Probes have increased sensitivity for distinguishing DNA base-pair mismatches, and are commonly used for SNP genotyping assays.

 

Locked Nucleic Acids (LNAs) can be incorporated into dual-labeled probes (DLPs) [1–4]. Because LNA bases significantly increase Tm, LNA PrimeTime 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]. A DNA DLP typically has a ΔTm of ~5°C between perfect-match and mismatch hybridization. An LNA DLP 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.

 

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

Mini LNA PrimeTime® Probes
  • Ideal for analyzing a small sample set or performing a few reactions to optimize probe designs
  • Available with FAM and HEX dyes and Iowa Black FQ
  • Guaranteed normalized yield of 0.5 nmoles

 

LNA PrimeTime® Probes
  • Available with FAM, Cy3®, Cy5®, TEX, TYE, and HEX dyes
  • High synthesis scales (250 nmole and 1 µmole) for large-scale and high throughput requirements

 

Design Considerations
  • Depending on sequence context, insertion of an LNA base into a DNA oligo can increase the Tm by 3–6°C. However, there are some sequence-specific designs involving G•T and C•A mismatches where LNA bases actually impair specificity [1].
  • LNA 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 LNA bases can be added towards the 3′-end of the probe to adjust Tm as needed.
  • 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. In addition, IDT recommends up to 6 LNA bases be placed in an LNA DLP.

 

References

  • 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 Meth, 196:104-112. Standard desalted IDT Primers and ZEN Double-Quenched Probes were used for HCV detection and genotyping. The primers and probe used in this study were designed based on 45 HCV sequences (obtained from GenBank) correspond to genotypes 1a, 1b, 2a, 2b, 2c, 3a, 3b and 4 according to the HCV genotype nomenclature.The developed assays for HCV detection and genotyping were set up to detect and discriminate four HCV genotypes.
  • Owczarzy R, You Y, et al. (2011) Stability and mismatch of Locked Nucleic Acid–DNA duplexes. Biochemistry . Biochemistry, 50(43):9352–67.
  • Johnson MP, Haupt LM, and 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.
  • Ugozzoli LA, Latorra D, et al. (2004) Real-time genotyping with oligonucleotide probes containing locked nucleic acids. Analytical Biochemistry, 324(1):143–152.
  • Letertre C, Perelle S, et al. (2003) Evaluation of the performance of LNA and MGB probes in 5′-nuclease assays. Molecular and Cellular Probes. Molecular and Cellular Probes, 17(6):307–311.

 

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