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Automated Robotic Liquid Handling Assembly of Modular DNA Devices
Published on: December 1, 2017
Design and assembly of large synthetic DNA constructs
Aleksandr E Miklos1, Randall A Hughes, Andrew D Ellington
1The University of Texas at Austin, Applied Research Laboratories, Department of Chemistry and Biochemistry, Center for Systems and Synthetic Biology, Austin, Texas, USA.
Current Protocols in Molecular Biology
|August 9, 2012
Summary
In-house gene synthesis offers significant time and cost advantages for researchers. This guide details designing DNA sequences and assembling them using polymerase chain reaction (PCR) for efficient gene construction.
Area of Science:
- Molecular Biology
- Synthetic Biology
- Biotechnology
Background:
- Custom synthetic gene-length DNA products are vital for research, but commercial services can be costly.
- The assembly of synthetic oligonucleotides into large DNA constructs is feasible for in-house execution.
- In-house gene synthesis presents opportunities for time and cost savings in research.
Purpose of the Study:
- To provide a comprehensive guide for designing and assembling synthetic gene-length DNA constructs.
- To outline strategies for efficient in-house gene synthesis, reducing reliance on commercial services.
- To detail methods for troubleshooting gene assembly failures.
Main Methods:
- Design of DNA sequences optimized for synthesis.
- Development of overlapping oligonucleotide schemes for seamless assembly.
- Utilizing a series of polymerase chain reaction (PCR) reactions for construct assembly.
- Implementing a troubleshooting protocol for identifying assembly failures at the oligonucleotide level.
Main Results:
- Successful assembly of custom DNA constructs through strategic oligonucleotide design and PCR.
- Identification of specific oligonucleotide pairs causing assembly failures.
- Demonstration of facile assembly for challenging DNA sequences.
Conclusions:
- In-house gene synthesis is a viable and cost-effective alternative to commercial services.
- Effective design strategies and PCR-based assembly protocols facilitate successful gene construction.
- The provided troubleshooting method enhances the reliability of gene synthesis efforts.
