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Efficient Method for Molecular Characterization of the 5′ and 3′ Ends of the Dengue Virus Genome

Dengue is a mosquito-borne disease that is of major importance in public health. Although it has been extensively studied at the molecular level, sequencing of the 5′ and 3′ ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present stu...

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Autores principales: Rosales-Munar, Alicia, Alvarez-Diaz, Diego Alejandro, Laiton-Donato, Katherine, Peláez-Carvajal, Dioselina, Usme-Ciro, Jose A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290889/
https://www.ncbi.nlm.nih.gov/pubmed/32365696
http://dx.doi.org/10.3390/v12050496
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author Rosales-Munar, Alicia
Alvarez-Diaz, Diego Alejandro
Laiton-Donato, Katherine
Peláez-Carvajal, Dioselina
Usme-Ciro, Jose A.
author_facet Rosales-Munar, Alicia
Alvarez-Diaz, Diego Alejandro
Laiton-Donato, Katherine
Peláez-Carvajal, Dioselina
Usme-Ciro, Jose A.
author_sort Rosales-Munar, Alicia
collection PubMed
description Dengue is a mosquito-borne disease that is of major importance in public health. Although it has been extensively studied at the molecular level, sequencing of the 5′ and 3′ ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present study aimed to characterize the 5′ and 3′ ends of dengue virus types 1 to 4. The 5′ and 3′ ends of twenty-nine dengue virus isolates from acute infections were amplified through a modified protocol of the rapid amplification cDNA ends approach. For the 5′ end cDNA synthesis, specific anti-sense primers for each serotype were used, followed by polyadenylation of the cDNA using a terminal transferase and subsequent PCR amplification with oligo(dT) and internal specific reverse primer. At the 3′ end of the positive-sense viral RNA, an adenine tail was directly synthetized using an Escherichia coli poly(A) polymerase, allowing subsequent hybridization of the oligo(dT) during cDNA synthesis. The incorporation of the poly(A) tail at the 5′ and 3′ ends of the dengue virus cDNA and RNA, respectively, allowed for successful primer hybridization, PCR amplification and direct sequencing. This approach can be used for completing dengue virus genomes obtained through direct and next-generation sequencing methods.
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spelling pubmed-72908892020-06-17 Efficient Method for Molecular Characterization of the 5′ and 3′ Ends of the Dengue Virus Genome Rosales-Munar, Alicia Alvarez-Diaz, Diego Alejandro Laiton-Donato, Katherine Peláez-Carvajal, Dioselina Usme-Ciro, Jose A. Viruses Article Dengue is a mosquito-borne disease that is of major importance in public health. Although it has been extensively studied at the molecular level, sequencing of the 5′ and 3′ ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present study aimed to characterize the 5′ and 3′ ends of dengue virus types 1 to 4. The 5′ and 3′ ends of twenty-nine dengue virus isolates from acute infections were amplified through a modified protocol of the rapid amplification cDNA ends approach. For the 5′ end cDNA synthesis, specific anti-sense primers for each serotype were used, followed by polyadenylation of the cDNA using a terminal transferase and subsequent PCR amplification with oligo(dT) and internal specific reverse primer. At the 3′ end of the positive-sense viral RNA, an adenine tail was directly synthetized using an Escherichia coli poly(A) polymerase, allowing subsequent hybridization of the oligo(dT) during cDNA synthesis. The incorporation of the poly(A) tail at the 5′ and 3′ ends of the dengue virus cDNA and RNA, respectively, allowed for successful primer hybridization, PCR amplification and direct sequencing. This approach can be used for completing dengue virus genomes obtained through direct and next-generation sequencing methods. MDPI 2020-04-29 /pmc/articles/PMC7290889/ /pubmed/32365696 http://dx.doi.org/10.3390/v12050496 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Rosales-Munar, Alicia
Alvarez-Diaz, Diego Alejandro
Laiton-Donato, Katherine
Peláez-Carvajal, Dioselina
Usme-Ciro, Jose A.
Efficient Method for Molecular Characterization of the 5′ and 3′ Ends of the Dengue Virus Genome
title Efficient Method for Molecular Characterization of the 5′ and 3′ Ends of the Dengue Virus Genome
title_full Efficient Method for Molecular Characterization of the 5′ and 3′ Ends of the Dengue Virus Genome
title_fullStr Efficient Method for Molecular Characterization of the 5′ and 3′ Ends of the Dengue Virus Genome
title_full_unstemmed Efficient Method for Molecular Characterization of the 5′ and 3′ Ends of the Dengue Virus Genome
title_short Efficient Method for Molecular Characterization of the 5′ and 3′ Ends of the Dengue Virus Genome
title_sort efficient method for molecular characterization of the 5′ and 3′ ends of the dengue virus genome
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290889/
https://www.ncbi.nlm.nih.gov/pubmed/32365696
http://dx.doi.org/10.3390/v12050496
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