Polymerase chain reaction
Locked and loaded: New applications for LNA in PCR
Locked nucleic acids (LNA) have recently emerged as a powerful new tool for molecular biologists, and interesting new uses for this technology continue to be developed. One of the most promising areas of application for LNA, in addition to antisense-mediated inhibition of mRNA translation and detection of microRNAs, is the use of LNA-based oligonucleotides as primers in PCR and DNA sequencing.
Originally developed by scientists associated with the Danish biotechnology company Exiqon, LNA has several unique structural features that enable an unprecedented increase in duplex melting temperature. Available for all four bases, the core of the LNA technology is a bicyclic ribose derivative that contains a bridging methylene group between O-2’ and C-4’, leading to a reduction in the entropy of duplex formation and therefore providing a high thermal stability of LNA-DNA and LNA-DNA duplexes.
In order to be effective, LNA-containing oligonucleotides should be designed according to sequence-dependent thermodynamic parameters. Depending upon the sequence context, LNA-enabled duplex stabilisation can either be entropic or enthalpic, highlighting the importance of careful primer design for the accurate prediction of duplex melting temperature.
To simplify the improvement of poorly-performing PCR primers through the use of LNA, a team around Raymond Peterson at Celedon Laboratories, together with collaborators at the University of Maryland Department of Chemistry and Biochemistry, carried out a study to generate positional design rules for the incorporation of LNA into DNA oligonucleotides. Recently released by Nucleic Acids Research as an advance online publication, this report describes the performance analysis (using cycle thresholds, or Ct) of various primers with different patterns of LNA incorporation.
The main finding of the study was that incorporation of LNA monomers into the 5’ region of primers significantly improved the performance of PCR primers. Interestingly, LNA bases placed in the middle or near the 3’ end did not result in better primers, in all likelihood due to the stabilisation of mispriming events. LNA incorporation at the 5’ end, on the other hand, appears to enhance stability without increasing mispriming. These positional design rules should be useful for the systematic generation of LNA-containing PCR primers.
In another study, investigators at the Danish Institute for Food and Veterinary Research (DFVF) in Copenhagen evaluated various probe chemistries and platforms in an effort to improve the detection limit of quantitative real-time PCR (qPCR). This ambition is particularly relevant for qPCR applications involving food or environmental samples with relatively low amounts of target pathogens that nonetheless must be detected with dependable accuracy.
The DFVF team was able to determine that in comparison with TaqMan and Scorpion probes, LNA probes were the most sensitive, with up to 4.0 lower Ct values in the detection of very low levels of Salmonella DNA in various samples. These results suggest that LNA-based probes could be useful tools in the development and validation of qPCR methods with more sensitive detection limits.
An additional LNA-based PCR is mutation detection. In acute myelogenous leukemia (AML) patients, the most common genetic anomaly involves mutations of the nucleoplasmin gene (NPM1). Due in part to the nature of NPM1 mutations, but also with an eye towards higher throughput analysis for clinical diagnostics, PCR-based detection methods would be preferable over direct sequencing analysis.
In a recent report in Leukemia, investigators at the Technical University of Dresden, in collaboration with the companies Chipron and TIB Molbiol, developed a novel method using LNA-containing oligonucleotides to suppress the PCR amplification of wildtype NPM1 alleles, causing amplification signals to reflect levels of mutant alleles only. This LNA-mediated ‘PCR clamping’ method is capable of reliably detecting NPM1 mutations present only in a small minority of sample cells, and may also be appropriate for similar PCR-based genotyping protocols.
Taken together, these reports underscore the utility of LNA-containing oligonucleotides in the development of novel PCR techniques, and suggest that LNA is now ready for prime time.
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