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In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae

BACKGROUND: The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of m...

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Autores principales: Oliveira, Raffael AC, Almeida, Ricardo VM, Dantas, Márcia DA, Castro, Felipe N, Lima, João Paulo MS, Lanza, Daniel CF
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4119202/
https://www.ncbi.nlm.nih.gov/pubmed/25030031
http://dx.doi.org/10.1186/1471-2105-15-243
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author Oliveira, Raffael AC
Almeida, Ricardo VM
Dantas, Márcia DA
Castro, Felipe N
Lima, João Paulo MS
Lanza, Daniel CF
author_facet Oliveira, Raffael AC
Almeida, Ricardo VM
Dantas, Márcia DA
Castro, Felipe N
Lima, João Paulo MS
Lanza, Daniel CF
author_sort Oliveira, Raffael AC
collection PubMed
description BACKGROUND: The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively. RESULTS: A phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM. CONCLUSION: In silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2105-15-243) contains supplementary material, which is available to authorized users.
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spelling pubmed-41192022014-08-03 In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae Oliveira, Raffael AC Almeida, Ricardo VM Dantas, Márcia DA Castro, Felipe N Lima, João Paulo MS Lanza, Daniel CF BMC Bioinformatics Research Article BACKGROUND: The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively. RESULTS: A phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM. CONCLUSION: In silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2105-15-243) contains supplementary material, which is available to authorized users. BioMed Central 2014-07-16 /pmc/articles/PMC4119202/ /pubmed/25030031 http://dx.doi.org/10.1186/1471-2105-15-243 Text en © Oliveira et al.; licensee BioMed Central Ltd. 2014 This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Oliveira, Raffael AC
Almeida, Ricardo VM
Dantas, Márcia DA
Castro, Felipe N
Lima, João Paulo MS
Lanza, Daniel CF
In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae
title In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae
title_full In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae
title_fullStr In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae
title_full_unstemmed In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae
title_short In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae
title_sort in silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in totiviridae
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4119202/
https://www.ncbi.nlm.nih.gov/pubmed/25030031
http://dx.doi.org/10.1186/1471-2105-15-243
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