LNA Application PCR Probes

Applications

Overview

LNA may be used to enhance:

• Real-Time PCR probes
• In situ hybridisation probes
• Primers for single, multiplex and allele specific PCR
• Capture probes for SNP genotyping
• Capture probes for expression analysis
• Probes to monitor exon skipping

LNA should be used in any hybridisation assay, which requires high specificity and/or reproducibility.

For instance…

The LNA modification suits perfectly to SNP detection.

• First, the reduction of the size of the probe increases the impact of one mismatch in the stability of the duplex probe/target.
• Also, by designing probes with an LNA moiety in front of the variable position it becomes possible to discriminate very efficiently the allelic variations. The mismatch would avoid the A helix structure stabilisation and then decrease the Tm considerably.

This modification increases the specificity of the probe but also its power of discrimination.

Advantages

Affinity

• LNA increases the thermal stability of duplexes due to its RNA-like structure. 
• LNA:LNA duplex formation constitutes the most stable Watson-Crick base pairing system.

Tm modulation

• Depending on their position along the sequence, LNA bases allow to reach the desired Tm level without losing specificity.
• Introduction of LNA allows for shorter probes while maintaining the same Tm.

Specificity

• LNA enhances hybridization performance relative to native DNA, RNA or phosphorothioate.
• LNA lowers experimental error rates due to better mismatch discrimination.  
• LNA improves signal-to-noise ratio.

Enzyme compatibility

• LNA shows increased resistance to certain exo- and endonucleases thus leading to biostability.
• DNA-LNA chimeras readily activate RNAse H.  

• LNA acts as a substrate for standard molecular biology enzymes: T4 PNK, T4 DNA ligase, DNA polymerases.

Simplicity

• LNA behaves like DNA, so it is easily transferable to DNA-based assays.
• LNA is highly soluble in water.
• LNA complies with oligonucleotide synthesis and analysis methods (QC, purification, etc).

• LNA exhibits the same salt dependence as DNA and RNA.