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Novel 5′/3′RACE Method for Amplification and Determination of Single-Stranded RNAs Through Double-Stranded RNA (dsRNA) Intermediates

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Abstract

To acquire the full-length sequences and to determine the 5′/3′ends of the RNA genomes and mRNA transcripts using the rapid amplification of cDNA ends (RACE) protocols—via cDNA or mRNA templates—are a great challenge. This 4-steps RNA-based RACE method uses different ways to determine the RNA ends through a double-stranded (ds) RNA intermediate (dsRNA-RACE). In the first step a complementary RNA strand is synthesised by Phi6 RNA replicase enzyme next to the template ssRNA forming a dsRNA intermediate. The following steps include adapter ligation, nucleic acid purification and two classical methods with minor modifications reverse transcription and polymerase chain reaction. The dsRNA-RACE protocol could be used in wide variety of ssRNA (cellular, viral, bacterial, etc.) templates in the field of microbiology and cellular biology and suitable for the amplification of full-length RNAs including the 5′/3′ends. This is a novel, expansively utilizable molecular tool with fewer disadvantages than the existing 5′/3′RACE approaches.

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Acknowledgments

This study was supported by grants from the Hungarian Scientific Research Fund (OTKA K83013 and OTKA/NKFIH K111615). The authors of this manuscript have no competing interests to any company or manufacture that influence the results and discussion of this paper.

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Authors and Affiliations

  1. Regional Laboratory of Virology, National Reference Laboratory of Gastroenteric Viruses, ÁNTSZ Regional Institute of State Public Health Service, Szabadság út 7, Pecs, 7623, Hungary

    Péter Pankovics, Ákos Boros & Gábor Reuter

Authors
  1. Péter Pankovics
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  2. Ákos Boros
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  3. Gábor Reuter
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Correspondence to Gábor Reuter.

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Pankovics, P., Boros, Á. & Reuter, G. Novel 5′/3′RACE Method for Amplification and Determination of Single-Stranded RNAs Through Double-Stranded RNA (dsRNA) Intermediates. Mol Biotechnol 57, 974–981 (2015). https://doi.org/10.1007/s12033-015-9889-7

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