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Published on: October 1, 2017
Torsionally constrained DNA for single-molecule assays: an efficient, ligation-free method
D Hern Paik1, Violet A Roskens, Thomas T Perkins
1JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO 80309, USA and Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
Nucleic Acids Research
|August 13, 2013
Summary
We developed a new, ligation-free method using polymerase chain reaction (PCR) to create torsionally constrained DNA. This simplified technique yields high amounts of constrained DNA for single-molecule studies.
Area of Science:
- Molecular Biology
- Biophysics
- Biochemistry
Background:
- Controlled twisting of DNA is crucial for studying DNA topology enzymes.
- Existing methods for creating torsionally constrained DNA are laborious and yield low amounts of usable molecules.
Purpose of the Study:
- To develop a simplified, high-yield protocol for producing torsionally constrained DNA.
- To enable wider adoption of single-molecule DNA topology studies.
Main Methods:
- A novel ligation-free procedure using polymerase chain reaction (PCR).
- Utilized two 400-base-pair double-stranded DNA 'megaprimers' labeled with biotin or digoxigenin.
- Employed an optical-trap-based DNA-overstretching assay for constraint measurement.
Main Results:
- Achieved a high yield of gel-purified DNA (approx. 500 ng/100 µl PCR reaction).
- Obtained a high yield (84%) of torsionally constrained DNA molecules.
- The new method results in a single labeled strand, unlike traditional methods.
Conclusions:
- The PCR-based protocol significantly simplifies the preparation of torsionally constrained DNA.
- This method increases the accessibility and efficiency of single-molecule DNA topology assays.
- Facilitates broader research into DNA-modifying enzymes and DNA mechanics.
