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Identifying RNA Recombination Events and Non-covalent RNA–RNA Interactions with the Molecular Colony Technique

  • Helena V. Chetverina
  • Alexander B. Chetverin
Part of the Methods in Molecular Biology book series (MIMB, volume 1240)

Abstract

Molecular colonies (also known under names nanocolonies, polonies, RNA or DNA colonies, PCR colonies) form when nucleic acids are amplified in a porous solid or semi-solid medium, such as a gel, which contains a system for the exponential multiplication of RNA or DNA. As an individual colony comprises many copies of a single molecule (a molecular clone), the method can be used for the detection, enumeration, and analysis of individual DNA or RNA molecules, including the products of such rare events as RNA recombinations. Here we describe protocols for the detection of RNA molecules by growing colonies of RNA (in a gel containing Qβ replicase, the RNA-dependent RNA polymerase of phage Qβ) or cDNA (in a gel containing the components of PCR), and visualizing them by hybridization with fluorescent probes directly in the gel, including in real time, or by hybridization with fluorescent or radioactive probes followed by transfer to a nylon membrane.

Key words

Molecular colonies Nanocolonies Polymerase colony Polonies RNA colonies PCR colonies Agarose gel Polyacrylamide gel Nucleic acid amplification Real-time PCR Qβ replicase 

Notes

Acknowledgements

This work was supported by the Russian Foundation for Basic Research and by the program ‘Molecular and Cell Biology’ of the Presidium of the Russian Academy of Sciences. The images of molecular colonies were obtained by Helena Chetverina, Marina Falaleeva, Damir Kopein, and Alexandra Kravchenko.

References

  1. 1.
    Chetverin AB, Chetverina HV, Munishkin AV (1991) On the nature of spontaneous RNA synthesis by Qβ replicase. J Mol Biol 222:3–9PubMedCrossRefGoogle Scholar
  2. 2.
    Chetverin AB, Chetverina HV (1997) Method for amplification of nucleic acids in solid media.US Patent 5,616,478Google Scholar
  3. 3.
    Chetverina HV, Chetverin AB (1993) Cloning of RNA molecules in vitro. Nucleic Acids Res 21:2349–2353PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Samatov TR, Chetverina HV, Chetverin AB (2005) Expressible molecular colonies. Nucleic Acids Res 33:e145PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354PubMedCrossRefGoogle Scholar
  6. 6.
    Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491PubMedCrossRefGoogle Scholar
  7. 7.
    Mitra RD, Church GM (1999) In situ localized amplification and contact replication of many individual DNA molecules. Nucleic Acids Res 27:e34PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Chetverin AB, Chetverina HV (2008) Molecular colony technique: a new tool for biomedical research and clinical practice. Prog Nucleic Acid Res Mol Biol 82:219–255PubMedCrossRefGoogle Scholar
  9. 9.
    Chetverina HV, Chetverin AB (2008) Nanocolonies: detection, cloning, and analysis of individual molecules. Biochemistry (Mosc) 73:1361–1387CrossRefGoogle Scholar
  10. 10.
    Chetverina EV, Chetverin AB (2010) Nanocolonies and diagnostics of oncological diseases associated with chromosomal translocations. Biochemistry (Mosc) 75:1667–1691CrossRefGoogle Scholar
  11. 11.
    Chetverin AB, Chetverina HV, Demidenko AA, Ugarov VI (1997) Nonhomologous RNA recombination in a cell-free system: evidence for a transesterification mechanism guided by secondary structure. Cell 88:503–513PubMedCrossRefGoogle Scholar
  12. 12.
    Chetverina HV, Demidenko AA, Ugarov VI, Chetverin AB (1999) Spontaneous rearrangements in RNA sequences. FEBS Lett 450:89–94PubMedCrossRefGoogle Scholar
  13. 13.
    Chetverin AB, Kopein DS, Chetverina HV, Demidenko AA, Ugarov VI (2005) Viral RNA-directed RNA polymerases use diverse mechanisms to promote recombination between RNA molecules. J Biol Chem 280:8748–8755PubMedCrossRefGoogle Scholar
  14. 14.
    Chetverina HV, Falaleeva MV, Chetverin AB (2004) Simultaneous assay of DNA and RNA targets in the whole blood using novel isolation procedure and molecular colony amplification. Anal Biochem 334:376–381PubMedCrossRefGoogle Scholar
  15. 15.
    Falaleeva MV, Chetverina HV, Kravchenko AV, Chetverin AB (2009) Use of nanocolonies to detect minimal residual disease in patients with leukemia t(8;21). Mol Biol (Mosk) 43:166–174CrossRefGoogle Scholar
  16. 16.
    Chetverin AB (2004) Replicable and recombinogenic RNAs. FEBS Lett 567:35–41PubMedCrossRefGoogle Scholar
  17. 17.
    Chetverin AB (2011) Paradoxes of replication of RNA of a bacterial virus. Mol Biol (Mosk) 45:127–137CrossRefGoogle Scholar
  18. 18.
    Steinschneider A, Fraenkel-Konrat H (1966) Studies of nucleotide sequences in tobacco mosaic virus nucleic acid. III. Periodat oxidation and semicarbazone formation. Biochemistry 5:2729–2734PubMedCrossRefGoogle Scholar
  19. 19.
    Chetverina HV, Samatov TR, Ugarov VI, Chetverin AB (2002) Molecular colony diagnostics: detection and quantitation of viral nucleic acids by in-gel PCR. Biotechniques 33:150–156PubMedGoogle Scholar
  20. 20.
    Temin HM (1993) Retrovirus variation and reverse transcription: abnormal strand transfers result in retrovirus genetic variation. Proc Natl Acad Sci U S A 90:6900–6903PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Negroni M, Riccheti M, Nouvel P, Buc H (1995) Homologous recombination by reverse transcriptase during copying of two distinct RNA templates. Proc Natl Acad Sci U S A 92:6971–6975PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Diaz L, DeStefano JJ (1996) Strand transfer is enhanced by mismatched nucleotides at the 3' primer terminus: a possible link between HIV reverse transcriptase fidelity and recombination. Nucleic Acids Res 24:3086–3092PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Samatov TR, Chetverina HV, Chetverin AB (2006) Real-time monitoring of DNA colonies growing in a polyacrylamide gel. Anal Biochem 356:300–302PubMedCrossRefGoogle Scholar
  24. 24.
    Chetverina EV, Kravchenko AV, Falaleeva MV, Chetverin AB (2007) Express hybridization of molecular colonies with fluorescent probes. Russ J Bioorg Chem 33:423–430CrossRefGoogle Scholar
  25. 25.
    Blumenthal T (1979) Qβ RNA replicase and protein synthesis elongation factors EF-Tu and EF-Ts. Methods Enzymol 60:628–638PubMedCrossRefGoogle Scholar
  26. 26.
    Berestowskaya NH, Vasiliev VD, Volkov AA, Chetverin AB (1988) Electron microscopy study of Qβ replicase. FEBS Lett 228:263–267PubMedCrossRefGoogle Scholar
  27. 27.
    Vasiliev NN, Jenner L, Yusupov MM, Chetverin AB (2010) Isolation and crystallization of a chimeric Qβ replicase containing Thermus thermophilus EF-Ts. Biochemistry (Mosc) 75:989–994CrossRefGoogle Scholar
  28. 28.
    Khandjian EW (1986) UV crosslinking of RNA to nylon membrane enhances hybridization signals. Mol Biol Rep 11:107–115PubMedCrossRefGoogle Scholar
  29. 29.
    Jones RW, Jones MJ (1992) Simplified filter paper sandwich blot provides rapid, background-free northern blots. Biotechniques 12:685–688Google Scholar
  30. 30.
    Gordeev AA, Samatov TR, Chetverina HV, Chetverin AB (2011) 2D format for screening bacterial cells at the throughput of flow cytometry. Biotechnol Bioeng 108:2682–2690PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Helena V. Chetverina
    • 1
  • Alexander B. Chetverin
    • 1
  1. 1.Institute of Protein Research of the Russian Academy of SciencesPushchinoRussia

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