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Related Concept Videos

DNA Isolation01:24

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DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
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A method for generating user-defined circular single-stranded DNA from plasmid DNA using Golden Gate intramolecular

Isabell K Strawn1, Paul J Steiner1, Matilda S Newton1

  • 1Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado, USA.

Biotechnology and Bioengineering
|June 27, 2023
PubMed
Summary

Researchers developed a new method for creating long circular single-stranded DNA (cssDNA) using Golden Gate assembly. This robust technique efficiently produces user-defined cssDNA for biotechnology applications.

Keywords:
Golden Gate assemblyIntramolecular ligationssDNA synthesis

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Area of Science:

  • Molecular Biology
  • Biotechnology

Background:

  • Synthesis of long single-stranded DNA (ssDNA) molecules, including circular (cssDNA) and linear (lssDNA) forms, is crucial for diverse biotechnological applications.
  • Existing methods often struggle to scale for producing multikilobase ssDNA constructs.

Purpose of the Study:

  • To develop a robust and scalable methodology for generating user-defined long circular single-stranded DNA (cssDNA).
  • To evaluate the potential for producing linear single-stranded DNA (lssDNA) from the synthesized cssDNA.

Main Methods:

  • Employed Golden Gate assembly, a nickase, and exonuclease degradation for cssDNA synthesis.
  • Demonstrated the technique on plasmids with insert sizes from 2.1 to 3.4 kilobases (kb).
  • Assessed CRISPR-Cas9 cleavage conditions for converting cssDNA to lssDNA.

Main Results:

  • Achieved a yield of 33%-43% of theoretical for cssDNA synthesis.
  • The method requires no specialized equipment and can be completed within 5 hours.
  • Reported a 52% ± 8% cleavage efficiency for cssDNA to lssDNA conversion using CRISPR-Cas9.

Conclusions:

  • The developed protocol provides a reliable method for producing user-defined long cssDNA, making it accessible for biotechnology research.
  • While efficient for cssDNA, the current method's lssDNA generation capability does not surpass existing protocols.