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

Overview

Journal Mol Biotechnol

Publisher Springer

Specialties Biotechnology
Molecular Biology

Date 2015 Aug 29

PMID 26315976

Citations 3

Authors

Peter Pankovics

Akos Boros

Gabor Reuter

Affiliations

Soon will be listed here.

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.

Citing Articles

The complete genome sequences of a negative single-stranded RNA virus and a double-stranded RNA virus coinfecting the entomopathogenic fungus Beauveria bassiana Vuillemin.

Xu M, Liu H, Jia X, Zou X, Lu Y, Sui L Arch Virol. 2024; 169(3):42.

PMID: 38332318 DOI: 10.1007/s00705-024-05985-w.

A novel mammarenavirus (family Arenaviridae) in hedgehogs (Erinaceus roumanicus) in Europe.

Reuter G, Boros A, Takats K, Matics R, Pankovics P Arch Virol. 2023; 168(7):174.

PMID: 37291370 PMC: 10250479. DOI: 10.1007/s00705-023-05804-8.

Whole-genome sequence analysis of a novel orthobunyavirus isolated in Japan in the 1980s.

Yanase T, Murota K, Suda Y Arch Virol. 2023; 168(2):67.

PMID: 36653513 DOI: 10.1007/s00705-022-05639-9.

References

Paschal B, McReynolds L, Noren C, Nichols N . RNA polymerases. Curr Protoc Mol Biol. 2008; Chapter 3:Unit3.8. DOI: 10.1002/0471142727.mb0308s84. View

Chenchik A, Diachenko L, Moqadam F, Tarabykin V, Lukyanov S, Siebert P . Full-length cDNA cloning and determination of mRNA 5' and 3' ends by amplification of adaptor-ligated cDNA. Biotechniques. 1996; 21(3):526-34. DOI: 10.2144/96213pf02. View

Pankovics P, Boros A, Reuter G . Novel picornavirus in domesticated common quail (Coturnix coturnix) in Hungary. Arch Virol. 2011; 157(3):525-30. DOI: 10.1007/s00705-011-1192-8. View

van Ooij M, Polacek C, Glaudemans D, Kuijpers J, van Kuppeveld F, Andino R . Polyadenylation of genomic RNA and initiation of antigenomic RNA in a positive-strand RNA virus are controlled by the same cis-element. Nucleic Acids Res. 2006; 34(10):2953-65. PMC: 1474053. DOI: 10.1093/nar/gkl349. View

Makeyev E, Bamford D . Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage phi6. EMBO J. 2000; 19(1):124-33. PMC: 1171784. DOI: 10.1093/emboj/19.1.124. View

Arezi B, Hogrefe H . Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase thermostability through tighter binding to template-primer. Nucleic Acids Res. 2008; 37(2):473-81. PMC: 2632894. DOI: 10.1093/nar/gkn952. View

Makeyev E, Grimes J . RNA-dependent RNA polymerases of dsRNA bacteriophages. Virus Res. 2004; 101(1):45-55. DOI: 10.1016/j.virusres.2003.12.005. View

Szymkowiak C, Kwan W, Su Q, Toner T, Shaw A, Youil R . Rapid method for the characterization of 3' and 5' UTRs of influenza viruses. J Virol Methods. 2002; 107(1):15-20. DOI: 10.1016/s0166-0934(02)00184-2. View

Wakuda M, Pongsuwanna Y, Taniguchi K . Complete nucleotide sequences of two RNA segments of human picobirnavirus. J Virol Methods. 2005; 126(1-2):165-9. DOI: 10.1016/j.jviromet.2005.02.010. View

10.

Alfson K, Beadles M, Griffiths A . A new approach to determining whole viral genomic sequences including termini using a single deep sequencing run. J Virol Methods. 2014; 208:1-5. DOI: 10.1016/j.jviromet.2014.07.023. View

11.

Huang J, Chen F . Simultaneous amplification of 5' and 3' cDNA ends based on template-switching effect and inverse PCR. Biotechniques. 2006; 40(2):187-9. DOI: 10.2144/000112051. View

12.

Yang H, Makeyev E, Bamford D . Comparison of polymerase subunits from double-stranded RNA bacteriophages. J Virol. 2001; 75(22):11088-95. PMC: 114688. DOI: 10.1128/JVI.75.22.11088-11095.2001. View

13.

Herbert Z, Pollak E, Zougman A, Boros A, Kapan N, Molnar L . Identification of novel neuropeptides in the ventral nerve cord ganglia and their targets in an annelid worm, Eisenia fetida. J Comp Neurol. 2009; 514(5):415-32. DOI: 10.1002/cne.22043. View

14.

Boros A, Pankovics P, Simmonds P, Reuter G . Novel positive-sense, single-stranded RNA (+ssRNA) virus with di-cistronic genome from intestinal content of freshwater carp (Cyprinus carpio). PLoS One. 2011; 6(12):e29145. PMC: 3241695. DOI: 10.1371/journal.pone.0029145. View

15.

Makeyev E, Bamford D . The polymerase subunit of a dsRNA virus plays a central role in the regulation of viral RNA metabolism. EMBO J. 2000; 19(22):6275-84. PMC: 305833. DOI: 10.1093/emboj/19.22.6275. View

16.

Zhuang F, Fuchs R, Robb G . Small RNA expression profiling by high-throughput sequencing: implications of enzymatic manipulation. J Nucleic Acids. 2012; 2012:360358. PMC: 3388297. DOI: 10.1155/2012/360358. View

17.

Ohara O, Dorit R, Gilbert W . One-sided polymerase chain reaction: the amplification of cDNA. Proc Natl Acad Sci U S A. 1989; 86(15):5673-7. PMC: 297692. DOI: 10.1073/pnas.86.15.5673. View

18.

Maruyama I, Rakow T, Maruyama H . cRACE: a simple method for identification of the 5' end of mRNAs. Nucleic Acids Res. 1995; 23(18):3796-7. PMC: 307285. DOI: 10.1093/nar/23.18.3796. View

19.

Rozovics J, Virgen-Slane R, Semler B . Engineered picornavirus VPg-RNA substrates: analysis of a tyrosyl-RNA phosphodiesterase activity. PLoS One. 2011; 6(3):e16559. PMC: 3049768. DOI: 10.1371/journal.pone.0016559. View

20.

Frohman M, Dush M, Martin G . Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988; 85(23):8998-9002. PMC: 282649. DOI: 10.1073/pnas.85.23.8998. View