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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...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2017
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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. |
format | Online Article Text |
id | pubmed-5238799 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
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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