Dual-Labeled LNA Probes

Locked Nucleic Acids (LNAs) can be incorporated into dual-labeled probes.^1,2,3 Since LNA bases significantly increase Tm, LNA dual-labeled probes (DLPs) are shorter than standard DNA DLPs. Shorter probes have better quenching, 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). A DNA DLP typically has a Tm of ~5°C between perfect match and mismatch hybridization. An LNA DLP can have a Tm of > 15°C, greatly increasing accuracy of allele determination in real time PCR or other methods that use differential hybridization to distinguish polymorphism.

Depending on sequence context, insertion of an LNA base into a DNA oligo can increase the Tm by 3-6°C. IDT recommends 6 LNA bases be placed in an LNA DLP. 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. Note that relative binding affinity (Tm) of LNA bases are LNA: LNA > LNA:DNA > DNA>DNA. It is important to examine the probe sequence for self-dimer and hairpin formation and minimize designs that allow for LNA:LNA pairing.

Prices below include synthesis of the custom oligo (up to 25 bases in length), up to 6 LNA base insertions, reporter, quencher and HPLC Purification. Nanomole yields are listed for Dual-labeled LNA Probes 10 to 30 bases in length. Turnaround time for Dual-labeled LNA Probes is 4-6 business days. Please contact technical support at 1-800-328-2661 for assistance with design on LNA DLPs.

Reporter: 5' TYE^TM 563

Reporter: 5' TYE^TM 665

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References

1. Evaluation of the performance of LNA and MGB probes in 5'-nuclease assays. Letertre, C., Perelle, S., Dilasser, F., Arar, K., and Fach, P. Molecular and Cellular Probes, 17:307-311 (2003).
2. Real-time genotyping with oligonucleotide probes containing locked nucleic acids. Ugozzoli, L.A., Latorra. D., Pucket, R., Arar, K., and Hamby, K. Analytical Biochemistry, 324:143-152 (2004).
3. Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR. Johnson, M.P., Haupt, L.M., and Griffiths, L.R. Nucleic Acids Res., 32:e55 (2004).