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Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks
Published on: November 25, 2015
Highly efficient deletion method for the engineering of plasmid DNA with single-stranded oligonucleotides
Lin-Yu Lu1, Michael S Y Huen, Andrew Chi-Pang Tai
1University of Hong Kong, Pokfulam, Hong Kong, SAR, China.
Biotechniques
|March 12, 2008
Summary
The lambda phage Red recombination system enables precise DNA modifications using single-stranded oligonucleotides (SSOs). Optimized SSO-mediated deletion allows efficient plasmid manipulation without selectable markers.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- The lambda phage Red recombination system offers precise DNA modification capabilities for plasmids, BACs, and chromosomal DNA.
- Previous studies identified factors influencing single base substitutions mediated by single-stranded oligonucleotides (SSOs).
Purpose of the Study:
- To investigate the mechanistic basis of SSO-mediated deletion on plasmids.
- To determine the efficiency and requirements for SSO-mediated deletion.
Main Methods:
- Utilized single-stranded oligonucleotides (SSOs) of varying lengths to mediate deletions in plasmid DNA.
- Assessed deletion efficiency and analyzed factors such as SSO homology length and host repair systems.
Main Results:
- SSOs as short as 63 bp could mediate deletions up to 2 kb with over 1% efficiency.
- Homology on both sides of the SSO flanking the deleted fragment was crucial for deletion.
- Lagging strand-oriented SSOs showed higher deletion efficiency, and the mismatch repair system did not significantly impact deletion rates.
Conclusions:
- SSO-mediated deletion is a versatile and efficient method for DNA manipulation.
- Optimized SSO design facilitates plasmid manipulation, potentially eliminating the need for selectable markers.
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