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 50 and 30 ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present stu...

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Autores:
Rosales Munar, Alicia
Álvarez Díaz, Diego Alejandro
Laiton Donato, Katherine
Peláez Carvajal, Dioselina
Usme Ciro, José Aldemar
Tipo de recurso:
Article of journal
Fecha de publicación:
2020
Institución:
Universidad Cooperativa de Colombia
Repositorio:
Repositorio UCC
Idioma:
OAI Identifier:
oai:repository.ucc.edu.co:20.500.12494/32699
Acceso en línea:
https://hdl.handle.net/20.500.12494/32699
Palabra clave:
Extremos de genoma
Virus dengue
RACE-PCR
Poli(A) polimerasa
Genome ends
Dengue virus
RACE-PCR
Poly(A) polymerase
Rights
openAccess
License
Atribución
Description
Summary: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 50 and 30 ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present study aimed to characterize the 50 and 30 ends of dengue virus types 1 to 4. The 50 and 30 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 50 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 30 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 50 and 30 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.