Multiplex enzymatic synthesis of DNA with single-base resolution

Area of Science:

• Biotechnology
• Molecular Biology
• Bioengineering

Background:

• Enzymatic DNA synthesis (EDS) uses enzymes and aqueous conditions, offering a user-friendly alternative to traditional chemical methods.
• Applications like protein engineering and spatial transcriptomics require high sequence diversity, necessitating adaptations in EDS for spatial control.

Purpose of the Study:

• To adapt enzymatic DNA synthesis for microscale spatial control.
• To achieve highly parallel DNA synthesis with single-base precision.

Main Methods:

• A novel synthesis cycle involving site-specific inkjet dispensing of enzymes and nucleotides.
• Microelectromechanical systems (MEMS) inkjet technology for precise enzyme and nucleotide delivery.
• Repeating synthesis cycles on immobilized DNA primers with bulk slide washing to remove blocking groups.

Main Results:

• Demonstrated microscale spatial control over nucleic acid sequence and length.
• Achieved highly parallel DNA synthesis with single-base control.
• Validated synthesis through hybridization assays and gel electrophoresis.

Conclusions:

• This work presents a significant advancement in enzymatic DNA synthesis by enabling spatial control at the microscale.
• The developed method facilitates the production of oligo pools and arrays with high sequence diversity for advanced biological applications.
• This technique offers a distinct approach for enzymatically synthesizing DNA in a parallel and precise manner.