Advertisement

Biochemistry (Moscow)

, Volume 83, Issue 7, pp 855–860 | Cite as

A Novel Fluorescent GFP Chromophore Analog-Based Dye for Quantitative PCR

  • A. A. Stakheev
  • D. Yu. Ryazantsev
  • Yu. K. Zvezdina
  • M. S. Baranov
  • S. K. Zavriev
Short Communications
  • 19 Downloads

Abstract

This is the first report describing the possibility of using a green fluorescent protein chromophore synthetic analog, P-HOBDI-BF2, as a fluorescent dye for a linear hydrolysis probe used in qPCR. The study was carried out on a system for detection of the plant pathogenic fungus Fusarium avenaceum using a plasmid containing translation elongation factor 1α fragment as a template. To estimate fluorogenic properties of P-HOBDI-BF2, 6-FAM-and BDP-FL-labeled probes were used. It was demonstrated that a synthetic dye based on the P-HOBDI-BF2 chromophore can be used for labeling hydrolysis probes for qPCR, but fluorescence increase levels for P-HOBDI-BF2-labeled probes were slightly lower than those for 6-FAM-labeled ones. At the same time, the sensitivity of P-HOBDI-BF2-based assays remained high, and this fact together with acceptable fluorescence levels suggests that this dye can be considered as an efficient alternative for reporters traditionally used for fluorescence detection in the FAM channel.

Keywords

quantitative PCR fluorophore probe green fluorescent protein fluorescence increase 

Abbreviations

BDP-FL

boron-dipyrromethene-based dye for fluorescence detection on FAM channel

BHQ1 and 2

black hole quenchers 1 and 2

6-FAM

6-carboxyfluorescein

GFP

green fluorescent protein

JOE

4′,5′-dichloro-2′,7′-dimethoxy-5(6)-carboxyfluorescein

P-HOBDI-BF2

((5Z)-5-(difluorobo-ryl-4-hydroxyphenyl)-methylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one)

qPCR

quantitative polymerase chain reaction

RBF

relative background fluorescence

RFA

relative fluorescence addition

RFI

relative fluorescence increase

RP-HPLC

reversed-phase high-performance liquid chromatography

TEF1α

translation elongation factor 1 alpha gene

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Kubista, M., Andrade, J. M., Bengtsson, M., Forootan, A., Joniak, J., Lind, K., Sindelka, R., Sjoback, R., Sjogreen, B., Strombom, L., Stahlberg, A., and Zoric, N. (2006) The real–time polymerase chain reaction, Mol. Aspects Med., 27, 95–125.CrossRefPubMedGoogle Scholar
  2. 2.
    Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggert, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandersompele, J., and Wittwer, C. T. (2009) The MIQE guidelines: minimum information for publication of quantitative real–time PCR experiments, Clin. Chem., 55, 611–622.CrossRefPubMedGoogle Scholar
  3. 3.
    Gasparic, M. B., Tengs, T., La Paz, J. L., Holst–Jensen, A., Pla, M., Esteve, T., Zei, J., and Gruden, K. (2010) Comparison of nine different real–time PCR chemistries for qualitative and quantitative applications in GMO detec–tion, Anal. Bioanal. Chem., 396, 2023–2029.CrossRefGoogle Scholar
  4. 4.
    Kralik, P., and Ricchi, M. (2017) A basic guide to real time PCR in microbial diagnostics: definitions, parameters, and everything, Front. Microbiol., 8, 108.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Maurin, M. (2012) Real–time PCR as a diagnostic tool for bacterial diseases, Expert Rev. Mol. Diagn., 12, 731–754.CrossRefPubMedGoogle Scholar
  6. 6.
    Maerle, A. V., Sergeev, I. V., and Alekseev, L. P. (2014) Immuno–PCR method: prospect of application, Immunology (Moscow), 1, 44–48.Google Scholar
  7. 7.
    Ryazantsev, D. Y., Voronina, D. V., and Zavriev, S. K. (2016) Immuno–PCR: achievements and perspectives, Biochemistry (Moscow), 81, 1754–1770.CrossRefGoogle Scholar
  8. 8.
    He, X., and Patfield, S. A. (2015) Immuno–PCR assay for sensitive detection of proteins in real time, Methods Mol. Biol., 1318, 139–148.CrossRefPubMedGoogle Scholar
  9. 9.
    Niemeyer, C. M., Adler, M., and Wacker, R. (2007) Detecting antigens by quantitative immuno–PCR, Nat. Protoc., 2, 1918–1930.CrossRefPubMedGoogle Scholar
  10. 10.
    Sano, T., Smith, T. L., and Cantor, C. R. (1992) Immuno–PCR: very sensitive detection by means of specific anti–body–DNA conjugates, Science, 258, 120–122.CrossRefPubMedGoogle Scholar
  11. 11.
    Rebrikov, D. V., Samatov, G. A., Trofimov, D. Y., Semenov, P. A., Savilova, A. M., Kofiadi, I. A., and Abramov, D. D. (2009) Real–Time PCR [in Russian], BINOM, Laboratoriya Znanii, Moscow.Google Scholar
  12. 12.
    Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H. (1991) Detection of specific polymerase chain reaction product by utilizing the 5′–3′ exonuclease activity of Thermus aquaticus DNA polymerase, Proc. Natl. Acad. Sci. USA, 88, 7276–7280.CrossRefPubMedGoogle Scholar
  13. 13.
    Crockett, A. O., and Wittwer, C. T. (2001) Fluorescein–labeled oligonucleotides for real–time PCR: using the inherent quenching of deoxyguanosine nucleotides, Anal. Biochem., 290, 89–97.CrossRefPubMedGoogle Scholar
  14. 14.
    Wang, G., Becker, E., and Mesa, C. (2007) Optimization of 6–carboxy–X–rhodamine concentration for real–time poly–merase chain reaction using molecular beacon chemistry, Can. J. Microbiol., 53, 391–397.CrossRefPubMedGoogle Scholar
  15. 15.
    Kurata, S., Kanagawa, T., Yamada, K., Torimura, M., Yokomaku, T., Kamagata, Y., and Kurane, R. (2001) Fluorescent quenching–based quantitative detection of spe–cific DNA/RNA using a BODIPY® FL–labeled probe or primer, Nucleic Acids Res., 29, e34.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Baranov, M. S., Lukyanov, K. A., Borissova, A. O., Shamir, J., Kosenkov, D., Slipchenko, L. V., Tolbert, L. M., Yampolsky, I. V., and Solntsev, K. M. (2012) Conformationally locked chromophores as models of excit–ed–state proton transfer in fluorescent proteins, J. Am. Chem. Soc., 134, 6025–6032.CrossRefPubMedGoogle Scholar
  17. 17.
    Stakheev, A. A., Ryazantsev, D. Yu., Gagkaeva, T. Yu., and Zavriev, S. K. (2011) PCR detection of Fusarium fungi with similar profiles of the produced mycotoxins, Food Control, 22, 462–468.CrossRefGoogle Scholar
  18. 18.
    Ryazantsev, D. Y., Kvach, M. V., Tsybulsky, D. A., Prokhorenko, I. A., Stepanova, I. A., Martynenko, Y. V., Gontarev, S. V., Shmanai, V. V., Zavriev, S. K., and Korshun, V. A. (2014) Design of molecular beacons: 3′–couple quenchers improve fluorogenic properties of a probe in real–time PCR assay, Analyst, 139, 2867–2872.CrossRefPubMedGoogle Scholar
  19. 19.
    Ryazantsev, D. Y., Tsybulsky, D. A., Prokhorenko, I. A., Kvach, M. V., Martynenko, Y. V., Philipchenko, P. M., Shmanai, V. V., Korshun, V. A., and Zavriev, S. K. (2012) Two–dye and one–or two–quencher DNA probes for real–time PCR assay: synthesis and comparison with TaqMan™ probe, Anal. Bioanal. Chem., 404, 59–68.CrossRefPubMedGoogle Scholar
  20. 20.
    Tsybulsky, D. A., Kvach, M. V., Ryazantsev, D. Y., Aparin, I. O., Stakheev, A. A., Prokhorenko, I. A., Martynenko, Y. V., Gontarev, S. V., Formanovsky, A. A., Zatsepin, T. S., Shmanai, V. V., Korshun, V. A., and Zavriev, S. K. (2016) Molecular beacons with JOE dye: influence of linker and 3′–couple quencher, Mol. Cell. Probes, 30, 285–290.CrossRefPubMedGoogle Scholar
  21. 21.
    Frizler, M., Yampolsky, I. V., Baranov, M. S., Stirnberg, M., and Gutschow, M. (2013) Chemical introduction of the green fluorescence: imaging of cysteine cathepsins by an irreversibly locked GFP fluorophore, Org. Biomol. Chem., 11, 5913–5921.CrossRefPubMedGoogle Scholar
  22. 22.
    Tsybulsky, D. A., Kvach, M. V., Stepanova, I. A., Korshun, V. A., and Shmanai, V. V. (2012) 4′,5′–Dichloro–2′,7′–dimethoxy–5(6)–carboxyfluorescein (JOE): synthesis and spectral properties of oligonucleotide conjugates, J. Org. Chem., 77, 977–984.CrossRefPubMedGoogle Scholar
  23. 23.
    Ryabinin, V. A., Kostina, E. V., and Sinyakov, A. N. (2017) Unexpected transformation of black hole quenchers in electrophoretic purification of the fluorescein–containing TaqMan probes, Nucleosides Nucleotides Nucleic Acids, 36, 418–427.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2018

Authors and Affiliations

  • A. A. Stakheev
    • 1
  • D. Yu. Ryazantsev
    • 1
  • Yu. K. Zvezdina
    • 1
  • M. S. Baranov
    • 1
  • S. K. Zavriev
    • 1
  1. 1.Shemyakin and Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia

Personalised recommendations