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Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation

Padlock probes are single-stranded DNA molecules that are circularized upon hybridization to their target sequence by a DNA ligase. In the following, the circulated padlock probes are amplified and detected with fluorescently labeled probes complementary to the amplification product. The hallmark of...

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Autores principales: Schneider, Nils, Meier, Matthias
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5238799/
https://www.ncbi.nlm.nih.gov/pubmed/27879431
http://dx.doi.org/10.1261/rna.057836.116
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author Schneider, Nils
Meier, Matthias
author_facet Schneider, Nils
Meier, Matthias
author_sort Schneider, Nils
collection PubMed
description Padlock probes are single-stranded DNA molecules that are circularized upon hybridization to their target sequence by a DNA ligase. In the following, the circulated padlock probes are amplified and detected with fluorescently labeled probes complementary to the amplification product. The hallmark of padlock probe assays is a high detection specificity gained by the ligation reaction. Concomitantly, the ligation reaction is the largest drawback for a quantitative in situ detection of mRNAs due to the low affinities of common DNA or RNA ligases to RNA–DNA duplex strands. Therefore, current protocols require that mRNAs be reverse transcribed to DNA before detection with padlock probes. Recently, it was found that the DNA ligase from Paramecium bursaria Chlorella virus 1 (PBCV-1) is able to efficiently ligate RNA-splinted DNA. Hence, we designed a padlock probe assay for direct in situ detection of mRNAs using the PBCV-1 DNA ligase. Experimental single-cell data were used to optimize and characterize the efficiency of mRNA detection with padlock probes. Our results demonstrate that the PBCV-1 DNA ligase overcomes the efficiency limitation of current protocols for direct in situ mRNA detection, making the PBCV-1 DNA ligase an attractive tool to simplify in situ ligation sequencing applications.
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spelling pubmed-52387992018-02-01 Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation Schneider, Nils Meier, Matthias RNA Method Padlock probes are single-stranded DNA molecules that are circularized upon hybridization to their target sequence by a DNA ligase. In the following, the circulated padlock probes are amplified and detected with fluorescently labeled probes complementary to the amplification product. The hallmark of padlock probe assays is a high detection specificity gained by the ligation reaction. Concomitantly, the ligation reaction is the largest drawback for a quantitative in situ detection of mRNAs due to the low affinities of common DNA or RNA ligases to RNA–DNA duplex strands. Therefore, current protocols require that mRNAs be reverse transcribed to DNA before detection with padlock probes. Recently, it was found that the DNA ligase from Paramecium bursaria Chlorella virus 1 (PBCV-1) is able to efficiently ligate RNA-splinted DNA. Hence, we designed a padlock probe assay for direct in situ detection of mRNAs using the PBCV-1 DNA ligase. Experimental single-cell data were used to optimize and characterize the efficiency of mRNA detection with padlock probes. Our results demonstrate that the PBCV-1 DNA ligase overcomes the efficiency limitation of current protocols for direct in situ mRNA detection, making the PBCV-1 DNA ligase an attractive tool to simplify in situ ligation sequencing applications. Cold Spring Harbor Laboratory Press 2017-02 /pmc/articles/PMC5238799/ /pubmed/27879431 http://dx.doi.org/10.1261/rna.057836.116 Text en © 2017 Schneider and Meier; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Method
Schneider, Nils
Meier, Matthias
Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation
title Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation
title_full Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation
title_fullStr Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation
title_full_unstemmed Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation
title_short Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation
title_sort efficient in situ detection of mrnas using the chlorella virus dna ligase for padlock probe ligation
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5238799/
https://www.ncbi.nlm.nih.gov/pubmed/27879431
http://dx.doi.org/10.1261/rna.057836.116
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