Archives of Virology

, Volume 106, Issue 3–4, pp 335–340 | Cite as

Sequence analysis of minute amounts of viroid RNA using the polymerase chain reaction (PCR)

  • H. Puchta
  • H. L. Sänger
Rapid Communication


The amount of viroid RNA required for sequence analysis can be reduced by five to six orders of magnitude when a modified “polymerase chain reaction” (PCR) is used for the amplification of the reversely transcribed, overlapping viroid cDNAs. By applying this procedure it is possible to establish the molecular structure also of those viroids which are present only in extremely low amounts in various crop plants and ornamentals.


Polymerase Chain Reaction Infectious Disease Sequence Analysis Molecular Structure Crop Plant 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. 1.
    Collasius M, Falk H, Ciesler C, Valet G (1989) How to build an inexpensive cyclotherm instrument for automated polymerase chain reaction. Anal Biochem 180, in pressGoogle Scholar
  2. 2.
    Diener T (ed) (1987) The viroids. Plenum Press, New YorkGoogle Scholar
  3. 3.
    Duran-Vila N, Roistacher CN, Rivera-Bustamante R, Semancik JS (1988) A definition of citrus viroid groups and their relationship to the exocortis disease. J Gen Virol 69: 3069–3080Google Scholar
  4. 4.
    Garcia-Arenal F, Pallas V, Flores R (1987) The sequence of a viroid from grapevine closely related to severe isolates of citrus exocortis viroid. Nucleic Acids Res 15: 4203–4210Google Scholar
  5. 5.
    Krawetz SA, Pon RT, Dixon GH (1989) Increased efficiency of the Taq polymerase catalyzed polymerase chain reaction. Nucleic Acids Res 17: 819Google Scholar
  6. 6.
    Ohno T, Takamatsu N, Meshi T, Okada Y (1983) Hop stunt viroid: molecular cloning and nucleotide sequence of the complete cDNA copy. Nucleic Acids Res 11: 6185–6197Google Scholar
  7. 7.
    Perkin Elmer Cetus (1988) PCR protocol. Norwalk, CT, U.S.A.Google Scholar
  8. 8.
    Puchta H, Ramm K, Hadas R, Bar-Joseph M, Luckinger R, Freimüller K, Sänger HL (1989) Nucleotide sequence of a hop stunt viroid (HSVd) isolate from grapefruit in Israel. Nucleic Acids Res 17: 1247Google Scholar
  9. 9.
    Puchta H, Ramm K, Luckinger R, Freimüller K, Sänger HL (1989) Nucleotide sequence of a hop stunt viroid (HSVa) isolate from the German grapevine rootstock 5 BB as determined by PCR-mediated sequence analyses. Nucleic Acids Res, submittedGoogle Scholar
  10. 10.
    Puchta H, Ramm K, Sänger HL (1988) Nucleotide sequence of a hop stunt viroid from the German grapevine cultivar “Riesling”. Nucleic Acids Res 16: 2730Google Scholar
  11. 11.
    Puchta H, Ramm K, Sänger HL (1988) The molecular structure of hop latent viroid (HLV), a new viroid occurring worldwide in hops. Nucleic Acids Res 16: 4197–4216Google Scholar
  12. 12.
    Puchta H, Ramm K, Sänger HL (1988) Molecular and biological properties of a cloned and infectious new sequence variant of cucumber pale fruit viroid (CPFV). Nucleic Acids Res 16: 8171Google Scholar
  13. 13.
    Puchta H, Sänger HL (1988) An improved procedure for the rapid one-step-cloning of full-length viroid cDNA. Arch Virol 101: 137–140Google Scholar
  14. 14.
    Rezaian MA, Koltunow AM, Krake LR (1988) Isolation of three viroids and a circular RNA from grapevines. J Gen Virol 69: 413–422Google Scholar
  15. 15.
    Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of β-globulin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350–1354Google Scholar
  16. 16.
    Sano T, Hataya T, Shikata E (1988) Complete nucleotide sequence of a viroid isolated from Etrog citron, a new member of hop stunt viroid group. Nucleic Acids Res 16: 347Google Scholar
  17. 17.
    Sano T, Hataya T, Terai Y, Shikata E (1986) Association of a viroid-like RNA from Plum Dapple Disease occurring in Japan. Proc Japan Acad 62B: 98–101Google Scholar
  18. 18.
    Sano T, Ohshima K, Hataya T, Uyeda I, Shikata E, Chou TG, Meshi T, Okada Y (1986) A viroid resembling hop stunt viroid in grapevines from Europe, the United States and Japan. J Gen Virol 67: 1673–1678Google Scholar
  19. 19.
    Schumacher J, Randles JW, Riesner D (1983) A two-dimensional electrophoretic technique for the detection of circular viroids and virusoids. Anal Biochem 135: 288–295Google Scholar
  20. 20.
    Semancik JS (ed) (1987) Viroids and viroid-like pathogens. CRC Press, Boca Raton, FloridaGoogle Scholar
  21. 21.
    Tabler M, Günther I, Kern R, Sänger HL (1989) A microscale procedure for isolating and sequencing the viroid RNA present in one gram of infected leaf tissue. J Virol Methods 23: 111–126Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • H. Puchta
    • 1
  • H. L. Sänger
    • 1
  1. 1.Abteilung ViroidforschungMax-Planck-Institut für BiochemieMartinsried bei MünchenGermany

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