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Programmed assembly of long DNA synthons: design, mechanism, and online monitoring

  • Veronika V. Shchur
  • Yuliya P. Burankova
  • Michail A. Shapira
  • Dmitry V. Klevzhits
  • Sergei A. UsanovEmail author
  • Aleksei V. YantsevichEmail author
Methods and protocols
  • 46 Downloads

Abstract

Synthesis of custom de novo DNA sequences is highly demanded by fast-growing field of synthetic biology. Usually DNA sequences with length more than 1 kb are assembled from smaller synthetic DNA fragments (synthons) obtained by PCR assembly. The ability to synthesize longer synthons sufficiently reduces efforts and time for DNA synthesis. We developed a novel rational oligonucleotide design and programmed approach for the assembly of synthetic DNA synthons up to 1550 bp. The developed procedure was thoroughly investigated by synthesis of cholesterol oxidase gene from Streptomyces lavendulae (1544 bp). Our approach is based on combined design, oligonucleotide concentration gradient, and specialized assembly program that directs assembly reaction to full-length gene in a stepwise manner. The process includes conventional thermodynamically balanced assembly, thermodynamically balanced inside-out elongation, and further amplification. The ability of DNA polymerase to perform programmed assembly is highly influenced by the presence of 5′ → 3′-exonuclease activity. Oligonucleotide probing of PCR assembly products allowed us to shed light on the nature of high molecular weight spurious by-products and to understand the mechanism of their formation. For the first time, we applied light scattering techniques for tracking of oligonucleotide annealing, analysis of gene assembly products, and even for real-time monitoring of gene assembly process.

Keywords

Gene synthesis Concatemer Dynamic light scattering Polymerase chain reaction Cholesterol oxidase 

Notes

Funding information

This work is funded by Separate Project from National Academy of Sciences of Belarus.

Compliance with ethical standards

Research involving human participants and/or animals

The authors declare that that work did not require human and/or animal participation.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2019_10099_MOESM1_ESM.pdf (525 kb)
ESM 1 (PDF 809 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Bioorganic ChemistryNational Academy of Sciences of BelarusMinskBelarus

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