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Molecular Biology

, Volume 52, Issue 6, pp 854–864 | Cite as

Preparation of Modified Combinatorial DNA Libraries via Emulsion PCR with Subsequent Strand Separation

  • S. A. LapaEmail author
  • K. S. Romashova
  • M. A. Spitsyn
  • V. E. Shershov
  • V. E. Kuznetsova
  • T. O. Guseinov
  • O. A. Zasedateleva
  • S. P. Radko
  • E. N. Timofeev
  • A. V. Lisitsa
  • A. V. Chudinov
GENOMICS. TRANSCRIPTOMICS
  • 24 Downloads

Abstract

A modification of the enzymatic method for the preparation of combinatorial random DNA libraries, which combines amplification in isolated microvolumes with the simultaneous incorporation of modified nucleotides and subsequent separation of DNA strands, was developed. Deoxyuridine triphosphate with hydrophobic substituents such as structural analogues of amino acid side chains in the C5 position of the pyrimidine ring was used to introduce modifications into DNA. To prevent competitive amplification, which reduces the representativeness of combinatorial libraries, PCR in inverse emulsion was used. The separation of the strands of PCR products was carried out. There were six single-stranded DNA libraries with complete substitution of deoxythymidine via modified analogues with various functional groups. These DNA libraries are suitable for generating aptamers to protein targets through additional hydrophobic interactions from the introductions of appropriate modifications, and are completely compatible with the SELEX aptamer selection methodology.

Keywords:

combinatorial DNA libraries modified nucleotides 2'-deoxyuridine-5'-triphosphate PCR in inverse emulsion DNA polymerases modified aptamers 

Notes

REFERENCES

  1. 1.
    Imaizumi Y., Kasahara Y., Fujita H., Kitadume S., Ozaki H., Endoh T., Kuwahara M., Sugimoto N. 2013. Efficacy of base-modification on target binding of small molecule DNA aptamers. J. Am. Chem. Soc. 135, 9412–9419.CrossRefGoogle Scholar
  2. 2.
    Lapa S.A., Chudinov A.V., Timofeev E.N. 2016. The toolbox for modified aptamers. Mol. Biotechnol. 58, 79–92.CrossRefGoogle Scholar
  3. 3.
    Baccaro A., Steck A.L., Marx A. 2012. Barcoded nucleotides. Angew. Chem. Int. Ed. Engl. 51, 254–257.CrossRefGoogle Scholar
  4. 4.
    Tuerk C., Gold L. 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 249, 505–510.CrossRefGoogle Scholar
  5. 5.
    Gold L., Ayers D., Bertino J., Bock C., Bock A., Brody E.N., Carter J., Dalby A.B., Eaton B.E., Fitzwater T., Flather D., Forbes A., Foreman T., Fowler C., Gawande B., et al. 2010. Aptamer-based multiplexed proteomic technology for biomarker discovery. PLoS One. 5, e15004.CrossRefGoogle Scholar
  6. 6.
    Rohloff J.C., Gelinas A.D., Jarvis T.C., Ochsner U.A., Schneider D.J., Gold L., Janjic N. 2014. Nucleic acid ligands with protein-like side chains: Modified aptamers and their use as diagnostic and therapeutic agents. Mol. Ther. Nucl. Acids. 3, e201.CrossRefGoogle Scholar
  7. 7.
    Hottin A., Marx A. 2016. Structural insights into the processing of nucleobase-modified nucleotides by DNA polymerases. Acc. Chem. Res. 49, 418–427.CrossRefGoogle Scholar
  8. 8.
    Chudinov A.V., Kiseleva Y.Y., Kuznetsov V.E., Shershov V.E., Spitsyn M.A., Guseinov T.O., Lapa S.A., Timofeev E.A., Archakov A.I., Lisitsa A.V., Radko S.P., Zasedatelev A.S. 2017. Structural and functional analysis of biopolymers and their complexes: Enzymatic synthesis of high-modified DNA. Mol. Biol. (Moscow). 51 (3), 474–483.CrossRefGoogle Scholar
  9. 9.
    Ho S.P., Britton D.H., Stone B.A., Behrens D.L., Leffet L.M., Hobbs F.W., Miller J.A., Trainor G.L. 1996. Potent antisense oligonucleotides to human multidrug resistance-1 mRNA are rationally selected by mapping RNA-accessible sites with oligonucleotide libraries. Nucleic Acids Res. 24, 1901–1907.CrossRefGoogle Scholar
  10. 10.
    Shao K., Ding W., Wang F., Li H., Ma D., Wang H. 2011. Emulsion PCR: A high efficient way of PCR amplification of random DNA libraries in aptamer selection. PLoS One. 6, e24910.CrossRefGoogle Scholar
  11. 11.
    Schütze T., Rubelt F., Repkow J., Greiner N., Erdmann V.A., Lehrach H., Konthur Z., Glökler J. 2011. A streamlined protocol for emulsion polymerase chain reaction and subsequent purification. Anal. Biochem. 410, 155–157.CrossRefGoogle Scholar
  12. 12.
    Diehl F., Li M., He Y., Kinzler K.W., Vogelstein B., Dressman D. 2006. BEAMing: Single-molecule PCR on microparticles in water-in-oil emulsions. Nat. Methods. 3, 551–559.CrossRefGoogle Scholar
  13. 13.
    Zhu Z., Zhang W., Leng X., Zhang M., Guan Z., Lu J., Yang C.J. 2012. Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level. Lab. Chip. 12, 3907–3913.CrossRefGoogle Scholar
  14. 14.
    Nakano M., Komatsu J., Matsuura S., Takashima K., Katsura S., Mizuno A. 2003. Single-molecule PCR using water-in-oil emulsion. J. Biotechnol. 102, 117–124.CrossRefGoogle Scholar
  15. 15.
    Musyanovych A., Mailander V., Landfester K. 2005. Miniemulsion droplets as single molecule nanoreactors for polymerase chain reaction. Biomacromolecules. 6, 1824–1828.CrossRefGoogle Scholar
  16. 16.
    Williams R., Peisajovich S.G., Miller O.J., Magdassi S., Tawfik D.S., Griffiths A.D. 2006. Amplification of complex gene libraries by emulsion PCR. Nat. Methods. 3, 545–550.CrossRefGoogle Scholar
  17. 17.
    Leamon J.H., Link D.R., Egholm M., Rothberg J.M. 2006. Overview: Methods and applications for droplet compartmentalization of biology. Nat. Methods. 3, 541–543.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2018

Authors and Affiliations

  • S. A. Lapa
    • 1
    Email author
  • K. S. Romashova
    • 1
  • M. A. Spitsyn
    • 1
    • 2
  • V. E. Shershov
    • 1
  • V. E. Kuznetsova
    • 1
  • T. O. Guseinov
    • 1
    • 2
  • O. A. Zasedateleva
    • 1
  • S. P. Radko
    • 2
    • 3
  • E. N. Timofeev
    • 1
  • A. V. Lisitsa
    • 3
  • A. V. Chudinov
    • 1
    • 2
  1. 1.Engelhardt Institute of Molecular Biology, Russian Academy of SciencesMoscowRussia
  2. 2.IBMC-EcoBioPharm Ltd.MoscowRussia
  3. 3.Orekhovich Institute of Biomedical Chemistry, Russian Academy of SciencesMoscowRussia

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