Synthesis of long cDNA from viral RNA template()

Methods to make long and reliable cDNA from viral RNA template have been optimized. The conditions of the denaturation of the viral RNA template were most critical. For synthesis of the first DNA strand, the concentration of the primer and the presence of an RNase inhibitor were important. During th...

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Detalles Bibliográficos
Autores principales: Lenstra, J.A., De Groot, R.J., Jacobs, L., Kusters, J.G., Niesters, H.G.M., Van Der Zeijst, B.A.M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Published by Elsevier B.V. 1988
Materias:
Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135641/
https://www.ncbi.nlm.nih.gov/pubmed/3192156
http://dx.doi.org/10.1016/0735-0651(88)90017-9
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author Lenstra, J.A.
De Groot, R.J.
Jacobs, L.
Kusters, J.G.
Niesters, H.G.M.
Van Der Zeijst, B.A.M.
author_facet Lenstra, J.A.
De Groot, R.J.
Jacobs, L.
Kusters, J.G.
Niesters, H.G.M.
Van Der Zeijst, B.A.M.
author_sort Lenstra, J.A.
collection PubMed
description Methods to make long and reliable cDNA from viral RNA template have been optimized. The conditions of the denaturation of the viral RNA template were most critical. For synthesis of the first DNA strand, the concentration of the primer and the presence of an RNase inhibitor were important. During the synthesis of the second strand, the incubation temperature was found to have effect on the length of the transcripts. Application of our optimized conditions on coronaviral genomic RNA as template resulted in cDNA libraries with inserts in the range of 0.5–5 kb without a separate cDNA size selection. Furthermore, a convenient variant of the alcohol precipitation and the analysis of single-stranded DNA on neutral agarose gels are described.
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spelling pubmed-71356412020-04-08 Synthesis of long cDNA from viral RNA template() Lenstra, J.A. De Groot, R.J. Jacobs, L. Kusters, J.G. Niesters, H.G.M. Van Der Zeijst, B.A.M. Gene Anal Tech Article Methods to make long and reliable cDNA from viral RNA template have been optimized. The conditions of the denaturation of the viral RNA template were most critical. For synthesis of the first DNA strand, the concentration of the primer and the presence of an RNase inhibitor were important. During the synthesis of the second strand, the incubation temperature was found to have effect on the length of the transcripts. Application of our optimized conditions on coronaviral genomic RNA as template resulted in cDNA libraries with inserts in the range of 0.5–5 kb without a separate cDNA size selection. Furthermore, a convenient variant of the alcohol precipitation and the analysis of single-stranded DNA on neutral agarose gels are described. Published by Elsevier B.V. 1988 2002-12-11 /pmc/articles/PMC7135641/ /pubmed/3192156 http://dx.doi.org/10.1016/0735-0651(88)90017-9 Text en Copyright © 1988 Published by Elsevier B.V. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
spellingShingle Article
Lenstra, J.A.
De Groot, R.J.
Jacobs, L.
Kusters, J.G.
Niesters, H.G.M.
Van Der Zeijst, B.A.M.
Synthesis of long cDNA from viral RNA template()
title Synthesis of long cDNA from viral RNA template()
title_full Synthesis of long cDNA from viral RNA template()
title_fullStr Synthesis of long cDNA from viral RNA template()
title_full_unstemmed Synthesis of long cDNA from viral RNA template()
title_short Synthesis of long cDNA from viral RNA template()
title_sort synthesis of long cdna from viral rna template()
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135641/
https://www.ncbi.nlm.nih.gov/pubmed/3192156
http://dx.doi.org/10.1016/0735-0651(88)90017-9
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