Use of VisualOMP for LNA based designs and the benefits of an LNA approach

This makes LNA substitutions into PCR primers, probes and sequencing primers, a powerful method for increasing specificity and efficiency of reactions by increasing binding efficiency of the intended oligonucleotides (ref 2 and 3).

VisualOMP is unique in being able to accurately model the inclusion of modified bases into an oligonucleotide and can accurately predict the effect of these additions. The predictions have been experimentally confirmed by UV melting experiments under a wide range of temperatures, buffer mixes and oligonucleotide compositions to confirm the accuracy of these predictions.

Below are a few examples where LNA modification can improve assay performance and some images from the software.

PCR

LNA incorporation, particularly at the 5’ end of primers has been shown to increase the specificity and reaction kinetics of PCR reactions. Further benefits appear to be that shorter primers can be designed, which is vital for areas of high AT content or when experimental concerns require the primers to be placed in a given space. Additionally LNA primers tend to generate PCR reactions whereby the efficiency is not as significantly reduced in the presence of lower primer and polymerase concentrations, indicating that the LNA modification helps to recruit the primers and polymerase into a complex capable of extending primers with more efficiency than a standard DNA molecule.

VisualOMP provides visual representations of all interactions; note the 12 base pair modified primer has a TM of 710C.

Real-time PCR

LNA modifications can also be successfully included in real-time PCR probes, such as 5’ nuclease assays as shown below. The benefits are again increased specificity to the intended target, but that much shorter probes can be designed. This is vital when only a short space is available for probe placement but also has the advantage of bringing fluorophore and quencher much closer together, with concurrent improvements in signal to noise ratio. LNA molecules are particularly suited to SNP genotyping assays, where it is possible to generate assays with no cross talk between genotypes as the unintended probe will simply be unable to bind under the assay conditions.

5’ nuclease probe incorporating 2 LNA moieties

Sequencing

In the case of sequencing reactions LNA molecules have been shown to increase the Phred scores of sequences. Further, LNA moieties incorporated at the 5’ end of primers will reduce n-1 effects in primers since any primer lacking the modified base will be less likely to hybridise in the absence of the stabilising influence of the LNA substituion. This will need to be accurately modelled in the VisualOMP software to determine the effect of n-1 and also the presence of SNPs under the sequencing primer.

Example of identical sequence, the top image was a standard primer and the bottom featured 2 LNA modified bases.

VisualOMP

The VisualOMP software is capable of in silico PCR, real-time micro-array and sequencing assay design. Crucially all predictions are experimentally confirmed and all predictions take into account both buffer constituents but also the thermodynamics of a mixed population of oligonucleotides.

For further information and a web based demonstration please contact us at: AssayDesign@biogene.com

1. Mouritzen et al. Single nucleotide polymorphism genotyping using locked nucleic acid (LNA).Expert Rev Mol Diagn. 2003 Jan;3(1):27-38
2. Levin et al. Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers. Nucleic Acids Research Advance Access published online on October 28, 2006
3. Yong et al:Design of LNA probes that improve mismatch discrimination. Nucleic Acids Research 2006 34(8):