Twin-primer non-enzymatic DNA assembly: an efficient and accurate multi-part DNA assembly method

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Scarless multi-fragment DNA assembly for large plasmids is a significant challenge in synthetic biology.
• Automation in molecular biology necessitates streamlined, operator-independent DNA assembly methods.
• Non-enzymatic DNA assembly approaches are highly desirable for automated workflows.

Purpose of the Study:

• To optimize and operationalize Twin-Primer Assembly (TPA) as an enzyme-free DNA assembly method.
• To evaluate TPA's efficiency and fidelity for constructing large plasmids from multiple fragments.
• To provide a streamlined, non-enzymatic solution for DNA assembly in automated synthetic biology.

Main Methods:

• Developed and optimized Twin-Primer Assembly (TPA), a non-enzymatic method for DNA fragment ligation.
• Utilized polymerase chain reaction (PCR)-amplified DNA fragments for plasmid construction.
• Assessed TPA performance in assembling plasmids of varying sizes and fragment numbers.

Main Results:

• TPA successfully assembled a 7 kb plasmid from 10 fragments with approximately 80% fidelity.
• TPA assembled a 31 kb plasmid from five fragments with approximately 50% fidelity.
• TPA demonstrated scarless and sequence-independent DNA cloning capabilities.

Conclusions:

• Twin-Primer Assembly (TPA) is an effective non-enzymatic method for scarless, multi-fragment DNA assembly.
• TPA performance is comparable to existing in vitro DNA assembly methods, particularly for automation.
• TPA offers a valuable tool for synthetic biologists, especially in automated molecular biology applications.