High-yield purification of exceptional-quality, single-molecule DNA substrates

Authors

  • Yue Lu
  • Piero Bianco University of Nebraska Medical Center

DOI:

https://doi.org/10.14440/jbm.2021.350

Keywords:

column chromatography, DNA substrate, oligonucleotide, single-molecule, TSKgel DNA-stat

Abstract

Single-molecule studies involving DNA or RNA, require homogeneous preparations of nucleic acid substrates of exceptional quality. Over the past several years, a variety of methods have been published describing different purification methods but these are frustratingly inconsistent with variable yields even in the hands of experienced bench scientists. To address these issues, we present an optimized and straightforward, column-based approach that is reproducible and produces high yields of substrates or substrate components of exceptional quality. Central to the success of the method presented is the use of a non-porous anion exchange resin. In addition to the use of this resin, we encourage the optimization of each step in the construction of substrates. The fully optimized method produces high yields of a hairpin DNA substrate of exceptional quality. While this substrate is suitable for single-molecule, magnetic tweezer experiments, the described method is readily adaptable to the production of DNA substrates for the majority of single-molecule studies involving nucleic acids ranging in size from 70–15000 bp.

Author Biography

Piero Bianco, University of Nebraska Medical Center

Res. Associate professor, Department of Pharmaceutical Sciences

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Published

2021-02-24

How to Cite

1.
Lu Y, Bianco P. High-yield purification of exceptional-quality, single-molecule DNA substrates. J Biol Methods [Internet]. 2021Feb.24 [cited 2021Dec.4];8(1):e145. Available from: https://jbmethods.org/jbm/article/view/350

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