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Enhanced detection and distinction of RNA by enzymatic probe ligation

Abstract

It is important that RNA molecules representing members of gene families are distinguished in expression analyses, and even greater resolving power may be required to identify allelic variants of transcripts in order to investigate imprinting or to study the distribution of mutant genes in tissues. Ligase-mediated gene detection allows precise distinction of DNA sequence variants1,2,3,4, but it is not known if ligases can also be used to distinguish variants of RNA sequences. Here we present conditions for efficient ligation of pairs of DNA oligonucleotides hybridizing next to one another on RNA strands, permitting discrimination of any single nucleotide probe–target mismatch by a factor of between 20- and 200-fold. The mechanism allows padlock probes to be used to distinguish single-nucleotide variants in RNA. Ligase-mediated gene detection could therefore provide highly sensitive and accurate ligase-mediated detection and distinction of RNA sequence variants in solution, on DNA microarrays, and in situ.

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Figure 1: Ligation probe oligonucleotides used in the study.
Figure 2: Ligation efficiency and target discrimination by pairs of oligonucleotide probes hybridized to RNA target molecules.
Figure 3: Ligase-mediated circularization of padlock probes is sensitive to single-nucleotide mismatches at the probe's 3′ end.

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Acknowledgements

We thank Andries Blokzij for excellent technical assistance. This work was supported by grants from the Beijer Foundation, the Swedish Medical and Technological Research Councils, and the Swedish Cancer Foundation. M.N. is supported by a long-term EMBO fellowship.

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Correspondence to Mats Nilsson.

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Nilsson, M., Barbany, G., Antson, DO. et al. Enhanced detection and distinction of RNA by enzymatic probe ligation . Nat Biotechnol 18, 791–793 (2000). https://doi.org/10.1038/77367

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