INTEGRATED<strong>DNA</strong>TECHNOLOGIESPRODUCTSFLUORESCENCE-BASED APPLICATIONSDual-Labeled <strong>DNA</strong> ProbesReporter 5’ TAMRA TM NHS EsterFreedom EquivalentQuencher Freedom Quencher Guaranteed Yields3’ Black Hole Quencher®-2 3’ Iowa Black® RQ 8 nm, 20 nmFreedom Quencher 5’ Iowa Black® FQFreedom ReporterGuaranteed Yields3’ 6-FAM TM 10 nm, 20 nm, 40 nm©2008 <strong>Integrated</strong> <strong>DNA</strong> <strong>Technologies</strong> www.idtdna.com53
INTEGRATED<strong>DNA</strong>TECHNOLOGIESPRODUCTSFLUORESCENCE-BASED APPLICATIONSDual-Labeled LNA ProbesLocked Nucleic Acids (LNAs) can be incorporated into dual-labeled probes. 1-3 Since LNA bases significantly increase Tm, LNADual-Labeled Probes (DLPs) are shorter than standard <strong>DNA</strong> DLPs. Shorter probes have better quenching, a higher signal-tonoiseratio and are more sensitive. More importantly, these probes offer an improved ability to distinguish mutations or singlenucleotide polymorphisms (SNPs). A <strong>DNA</strong> 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 methodsthat use differential hybridization to distinguish polymorphisms.Depending on sequence context, insertion of an LNA base into a <strong>DNA</strong> oligo can increase the Tm by 3-6°C. IDT recommends3-6 LNA bases be placed in an LNA DLP. LNA bases should be placed at the SNP site and adjacent bases. The SNP should bepositioned in the center of the probe if possible. Additional LNA bases can be added towards the 3’-end of the probe to adjustTm as needed. Note that relative binding affinity (Tm) of LNA bases is LNA:LNA > LNA:<strong>DNA</strong> > <strong>DNA</strong>:<strong>DNA</strong>. It is important toexamine 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, quencherand HPLC purification. Nanomole yields are listed for Dual-Labeled LNA Probes 10 to 25 bases in length. Turnaround time forDual-labeled LNA Probes is 4-6 business days. Please contact Customer Care at 800-328-2661 for assistance with the design ofLNA DLPs.Freedom Reporter5’ 6-FAM TMFreedom EquivalentQuencher Freedom Quencher Guaranteed Yields3’ Black Hole Quencher®-1 3’ Iowa Black® FQ 4 nm, 10 nmReporter 5’ Cy3 TMFreedom EquivalentQuencher Freedom Quencher Guaranteed Yields3’ Black Hole Quencher®-2 3’ Iowa Black® RQ 4 nm, 10 nmReporter 5’ Cy5 TMFreedom EquivalentQuencher Freedom Quencher Guaranteed Yields3’ Black Hole Quencher®-2 3’ Iowa Black® RQ 4 nm, 10 nmLocked Nucleic Acids are licensed from Exiqon, www.exiqon.com.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 CellularProbes, 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. AnalyticalBiochemistry, 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., andGriffiths, L.R. Nucleic Acids Res., 32:e55 (2004).©2008 <strong>Integrated</strong> <strong>DNA</strong> <strong>Technologies</strong> 54 www.idtdna.com
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