Please use this identifier to cite or link to this item:
|Title:||Efficient method for molecular characterization of the 5' and 3' ends of the dengue virus genome|
Usme Ciro, Jose Aldemar
|Keywords:||3' untranslated region|
5' untranslated region
high throughput sequencing
polymerase chain reaction
reverse transcription polymerase chain reaction
|Abstract:||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. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.|
|Publisher:||Multidisciplinary Digital Publishing Institute (MDPI)|
|Citation:||Rosales A,Alvarez DA,Laiton K,Peláez D,Usme JA. Efficient method for molecular characterization of the 5' and 3' ends of the dengue virus genome. Viruses. 2020. 12. (5):496. .|
|Appears in Collections:||Artículos Científicos|
Files in This Item:
There are no files associated with this item.
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.