Skip to main content
PMC home page
RNA logoLink to RNA
. 2001 May;7(5):774–781. doi: 10.1017/s1355838201002060

Primer-independent RNA sequencing with bacteriophage phi6 RNA polymerase and chain terminators.

E V Makeyev 1, D H Bamford 1
PMCID: PMC1370129  PMID: 11350041

Abstract

Here we propose a new general method for directly determining RNA sequence based on the use of the RNA-dependent RNA polymerase from bacteriophage phi6 and the chain terminators (RdRP sequencing). The following properties of the polymerase render it appropriate for this application: (1) the phi6 polymerase can replicate a number of single-stranded RNA templates in vitro. (2) In contrast to the primer-dependent DNA polymerases utilized in the sequencing procedure by Sanger et al. (Proc Natl Acad Sci USA, 1977, 74:5463-5467), it initiates nascent strand synthesis without a primer, starting the polymerization on the very 3'-terminus of the template. (3) The polymerase can incorporate chain-terminating nucleotide analogs into the nascent RNA chain to produce a set of base-specific termination products. Consequently, 3' proximal or even complete sequence of many target RNA molecules can be rapidly deduced without prior sequence information. The new technique proved useful for sequencing several synthetic ssRNA templates. Furthermore, using genomic segments of the bluetongue virus we show that RdRP sequencing can also be applied to naturally occurring dsRNA templates. This suggests possible uses of the method in the RNA virus research and diagnostics.

Full Text

The Full Text of this article is available as a PDF (777.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arens M. Methods for subtyping and molecular comparison of human viral genomes. Clin Microbiol Rev. 1999 Oct;12(4):612–626. doi: 10.1128/cmr.12.4.612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Attoui H., Billoir F., Cantaloube J. F., Biagini P., de Micco P., de Lamballerie X. Strategies for the sequence determination of viral dsRNA genomes. J Virol Methods. 2000 Sep;89(1-2):147–158. doi: 10.1016/s0166-0934(00)00212-3. [DOI] [PubMed] [Google Scholar]
  3. Axelrod V. D., Kramer F. R. Transcription from bacteriophage T7 and SP6 RNA polymerase promoters in the presence of 3'-deoxyribonucleoside 5'-triphosphate chain terminators. Biochemistry. 1985 Oct 8;24(21):5716–5723. doi: 10.1021/bi00342a005. [DOI] [PubMed] [Google Scholar]
  4. Blumenthal T. Q beta replicase template specificity: different templates require different GTP concentrations for initiation. Proc Natl Acad Sci U S A. 1980 May;77(5):2601–2605. doi: 10.1073/pnas.77.5.2601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Butcher S. J., Grimes J. M., Makeyev E. V., Bamford D. H., Stuart D. I. A mechanism for initiating RNA-dependent RNA polymerization. Nature. 2001 Mar 8;410(6825):235–240. doi: 10.1038/35065653. [DOI] [PubMed] [Google Scholar]
  6. Butcher S. J., Makeyev E. V., Grimes J. M., Stuart D. I., Bamford D. H. Crystallization and preliminary X-ray crystallographic studies on the bacteriophage phi6 RNA-dependent RNA polymerase. Acta Crystallogr D Biol Crystallogr. 2000 Nov;56(Pt 11):1473–1475. doi: 10.1107/s0907444900010702. [DOI] [PubMed] [Google Scholar]
  7. Clavijo A., Heckert R. A., Dulac G. C., Afshar A. Isolation and identification of bluetongue virus. J Virol Methods. 2000 Jun;87(1-2):13–23. doi: 10.1016/s0166-0934(00)00150-6. [DOI] [PubMed] [Google Scholar]
  8. Donis-Keller H., Maxam A. M., Gilbert W. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977 Aug;4(8):2527–2538. doi: 10.1093/nar/4.8.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eddy S. R. Noncoding RNA genes. Curr Opin Genet Dev. 1999 Dec;9(6):695–699. doi: 10.1016/s0959-437x(99)00022-2. [DOI] [PubMed] [Google Scholar]
  10. Gottlieb P., Strassman J., Qiao X., Frilander M., Frucht A., Mindich L. In vitro packaging and replication of individual genomic segments of bacteriophage phi 6 RNA. J Virol. 1992 May;66(5):2611–2616. doi: 10.1128/jvi.66.5.2611-2616.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hamlyn P. H., Browniee G. G., Cheng C. C., Gait M. J., Milstein C. Complete sequence of constant and 3' noncoding regions of an immunoglobulin mRNA using the dideoxynucleotide method of RNA sequencing. Cell. 1978 Nov;15(3):1067–1075. doi: 10.1016/0092-8674(78)90290-8. [DOI] [PubMed] [Google Scholar]
  12. Kim C. H., Kao C. C., Tinoco I., Jr RNA motifs that determine specificity between a viral replicase and its promoter. Nat Struct Biol. 2000 May;7(5):415–423. doi: 10.1038/75202. [DOI] [PubMed] [Google Scholar]
  13. Klovins J., van Duin J. A long-range pseudoknot in Qbeta RNA is essential for replication. J Mol Biol. 1999 Dec 10;294(4):875–884. doi: 10.1006/jmbi.1999.3274. [DOI] [PubMed] [Google Scholar]
  14. Kolb V. A., Makeyev E. V., Spirin A. S. Co-translational folding of an eukaryotic multidomain protein in a prokaryotic translation system. J Biol Chem. 2000 Jun 2;275(22):16597–16601. doi: 10.1074/jbc.M002030200. [DOI] [PubMed] [Google Scholar]
  15. Kramer F. R., Mills D. R. RNA sequencing with radioactive chain-terminating ribonucleotides. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5334–5338. doi: 10.1073/pnas.75.11.5334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Makeyev E. V., Bamford D. H. Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage phi6. EMBO J. 2000 Jan 4;19(1):124–133. doi: 10.1093/emboj/19.1.124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Makeyev E. V., Bamford D. H. The polymerase subunit of a dsRNA virus plays a central role in the regulation of viral RNA metabolism. EMBO J. 2000 Nov 15;19(22):6275–6284. doi: 10.1093/emboj/19.22.6275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mertens P. P., Sangar D. V. Analysis of the terminal sequences of the genome segments of four orbiviruses. Prog Clin Biol Res. 1985;178:371–387. [PubMed] [Google Scholar]
  20. Miller W. A., Bujarski J. J., Dreher T. W., Hall T. C. Minus-strand initiation by brome mosaic virus replicase within the 3' tRNA-like structure of native and modified RNA templates. J Mol Biol. 1986 Feb 20;187(4):537–546. doi: 10.1016/0022-2836(86)90332-3. [DOI] [PubMed] [Google Scholar]
  21. Patel P. H., Preston B. D. Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):549–553. doi: 10.1073/pnas.91.2.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Peattie D. A. Direct chemical method for sequencing RNA. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1760–1764. doi: 10.1073/pnas.76.4.1760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sasaki N., Izawa M., Watahiki M., Ozawa K., Tanaka T., Yoneda Y., Matsuura S., Carninci P., Muramatsu M., Okazaki Y. Transcriptional sequencing: A method for DNA sequencing using RNA polymerase. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3455–3460. doi: 10.1073/pnas.95.7.3455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zimmern D., Kaesberg P. 3'-terminal nucleotide sequence of encephalomyocarditis virus RNA determined by reverse transcriptase and chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4257–4261. doi: 10.1073/pnas.75.9.4257. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES