Locked nucleic acid is a promising and powerful technology for real-time quantitative polymerase chain reaction.
Locked nucleic acid (LNA) is a novel type of nucleic acid analogue that contains a 2'-O, 4'-C methylene bridge (Fig. 1). This bridge, locked in 3'-endo conformation, restricts the flexibility of the ribofuranose ring and locks the structure into a rigid bicyclic formation, conferring enhanced hybridisation performance and exceptional biological stability.
To overcome certain weaknesses of standard DNA probe chemistries, a new generation of sequence-specific, real-time quantitative polymerase chain reaction (PCR*) probes has been developed by custom oligonucleotide supplier, Proligo Primers & Probes.
Called LNA fluorescent probes, this new generation includes LNA LightCycler probes, LNA dual-labelled fluorogenic probes and LNA molecular beacons.
These probes provide a novel alternative in real-time PCR or end-point analytical assays for many applications such as gene expression profiling, mutation detection, allelic discrimination, single nucleotide polymorphisms (SNPs) and pathogen detection.
LNA fluorescent probes possess many advantages as described below.
Probe hybridisation
Introducing LNA chemistry into a real-time quantitative polymerase chain reaction (Q.PCR) probe increases thermal duplex stability and improves specificity of probe hybridisation to its target sequence.
As such, background fluorescence from spurious binding is reduced and the signal-to-noise ratio is increased.
The LNA monomer chemical structure enhances the stability of the hybridisation of the probe to its target.
As a result, the duplex melting temperature (Tm) may increase by up to 8°C per Locked nucleic acid monomer substitution in medium salt conditions (see table below), compared with a DNA fluorescent probe for the same target sequence, depending on the target nucleic acid.
This increase in hybridisation then creates a significant broadening in the scope of assay conditions and allows for more successful single-tube multiplexing.
The ability of Q.PCR probes to readily discriminate between SNPs, the most abundant form of genetic variation, is greatly enhanced by the incorporation of LNA bases.
Genotyping applications
Utilising LNA for allelic discrimination is an extremely reliable and effective means for SN-calling in genotyping applications.
The presence of a single base mismatch has a greater destabilising effect on the duplex formation between a LNA fluorescent probe and its target nucleic acid, than with a conventional DNA fluorescent probe.
Shorter probes
Shorter probes incorporating LNA bases can be used at the same temperatures as longer conventional DNA probes.
Due to Locked nucleic acid's enhanced hybridisation characteristics and then significant temperature contribution, LNA-containing Q.PCR probes can be synthesised to be shorter, allowing flexibility in design while still satisfying assay design guidelines.
As such, certain design limitations that cannot be overcome with standard DNA probe chemistries can now be reduced or eliminated.
In addition, by using LNA fluorescent probes, shorter probes can be designed to address traditionally problematic target sequences, such as AT- or GC-rich regions.
Also, the design of probes for querying difficult or inaccessible SNPs, such as the relatively stable G:T mismatch, is greatly facilitated by Locked nucleic acid.
For example, AT-rich Q.PCR probes often need to be over 30 bases long (and sometimes over 40 bases) to satisfy amplicon design guidelines but may still perform poorly.
Genotyping applications
With locked nucleic acid fluorescent probes, the selective placement of LNA base substitutions facilitates the optimal design of highly specific, shorter probes that perform very well, even at lengths of 13 to 20 bases!
LNA fluorescent probes are compatible with real-time PCR platforms and end-point analytical detection instruments, depending on the excitation/emission wavelengths of the dyes and the equipment.
Any instrumentation and reagents platform may be used under universal cycling conditions. No additional capital expenditure for specialised equipment is required.
Locked nucleic acid fluorescent probes offer recent and considerable advances in real-time polymerase chain reaction or end-point analytical assays for many applications notably in those dealing with superior thermal stability and hybridisation specificity. Affordable and easy to use, these probes are also suitable for high-throughput screening (HTS) assays.
Locked nucleic acid chemistry is expected to become the technique of choice across a wide range of research and diagnostic fields.
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PROLIGO SAS France, Paris, France. www.proligo.com. PCR (Polymerase Chain Reaction) is patented by Hoffman La Roche Ltd.
