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What do I need to consider when designing an LNA probe?

When designing LNA-containing oligonucleotides, one should follow the basic rules of primer design particularly pay attention to the location and number of LNAs.

For example, a typical 18-mer should contain a maximum of 7–8 LNAs.

Also, try to avoid stretches of more than 4 consecutive LNAs, which would result in very tight hybridization in that region.

Stretches of LNAs are to be avoided close to the 3′ end of an oligonucleotide. LNA will bind very tightly to other LNA residues. Avoid self-complementarity and cross-hybridization to other LNA-containing oligonucleotides.

Finally, be sure to match the Tm of the primers, as usual, keeping in mind that each substitution of a standard nucleotide with an LNA increases the Tm by 2-6 °C per LNA.

For novel applications, design guidelines may have to be established empirically by creating your own “best recipe”.