Custom Enzymatic DNA Synthesis Service
Principle of DNA Replication
DNA was found by the Swiss physician and biochemist Friedrich Miescher in human leucocytes in 1869. It is present in all prokaryotic and eukaryotic cells and many viruses. DNA codes genetic information for trait inheritance. The structure of the DNA molecule is highly stable. That makes it act as a template for the replication of new DNA molecules. A gene is a DNA segment responsible for synthesizing a specific protein within a cell. During DNA replication, the double-stranded DNA separates into two individual strands. Each strand acts as a template for the synthesis of a new complementary strand. The new strands are copied using hydrogen bonding between bases, similar to the principle observed in the double helix. Subsequently, two new double-stranded molecules of DNA are generated and each consists of the original strands and one new strand.
Fig.1 The structure of DNA.
Key Technologies
Our innovative approach is rooted in enzymatic DNA synthesis, a template-independent method that mimics nature's own processes. By precisely controlling the addition of individual nucleotides in a cyclical, step-by-step process, we can build custom DNA sequences with unprecedented accuracy and length.
Enzymatic Synthesis: Redefining DNA Customization
CD BioGlyco offers an enzymatic method for the synthesis of DNA based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) and nucleoside 5ʹ-triphosphates (NTPs). We utilize TdT to elongate oligonucleotides in the 5ʹ-to-3ʹ direction in a promiscuous manner. TdT is capable of accepting any of the four canonical nucleotides to generate diverse sequences simultaneously. To synthesize targeted sequences using TdT, a "reversible termination" mechanism is required to control the incorporation of nucleotides. In this mechanism, NTPs are modified with a synthesis-interrupting 'terminator' or protecting group (PG) at their 3ʹ position. This modification ensures the addition of one nucleotide per reaction step and is subsequently removed to allow for the incorporation of the next desired nucleotide.
- To achieve this, we utilize resin beads that contain pre-loaded chemically synthesized single-stranded initiator DNA. TdT then joins 3ʹ-protected NTPs onto this DNA, creating the desired sequence.
- After each elongation cycle, a washing step is employed to eliminate excess reagents and any side products.
- Additionally, at the end of each cycle, the removal of 3ʹ-PG blocking groups is carried out before the subsequent elongation cycle begins.
- The initiator DNA's 3ʹ end has a cleavage site with deoxyuridine that can be cleaved enzymatically. This site is cleaved by uracil DNA glycosylase upon the completion of the synthesis to release the assembled sequence.
- Upon completion of the synthesis, uracil DNA glycosylase cleaves this site to release the assembled sequence.
Fig.2 The repeat cycle steps of DNA synthesis. (CD BioGlyco)
Workflow
Publication Data
DOI.: 10.1093/nar/gkaf115
Journal: Nucleic Acids Research
IF: 13.1
Published: 2025
Results: This study details the directed evolution of a TdT for enzymatic DNA synthesis using 3'-phosphate-blocked dNTPs (3'P-dNTPs). Through 32 rounds of laboratory evolution introducing 80 amino acid substitutions (~20% of the protein sequence), the engineered TdT variant achieves >99% nucleotide coupling efficiency—a 200-fold improvement—while reducing extension times by >600-fold to 90 seconds per cycle. The evolved enzyme also exhibits enhanced thermostability (+20°C) and uniform activity across diverse oligonucleotide sequences and nucleotide types. Methodological refinements concretize an architecturally stable, linearly amplifiable biocatalyst executing high-fidelity oligonucleotide assembly within aqueous milieu, projecting this framework toward industrial enzymatic DNA manufacture.
Applications
- DNA can be used in the field of metabolic engineering. By utilizing parallel metabolic systems that interact with the natural cellular metabolic machinery, we can manipulate cells to perform practical tasks such as producing cost-effective chemicals or drugs through programming.
- By using the clarified DNA nucleotide sequence and amino acid sequence, we are able to synthesize polypeptides and protein genes that can be applied in actual production and research.
- DNA synthesis can be applied in medicine as DNA plays a key role as a valuable tool for clinical diagnosis.
Advantages
- Our method utilizes enzyme specificity and mild conditions to minimize side product formation and DNA damage, enabling direct synthesis of longer DNA without compromising quality.
- Our enzymatic synthesis reactions are environmentally friendly, promoting sustainability and minimizing ecosystem impact. Because they don't generate hazardous waste.
- Our strategy simplifies the synthesis process and streamlines the overall workflow.
- Our strategy utilizes enzyme engineering techniques to enhance the efficiency, specificity, and overall performance of our DNA synthesis process.
Frequently Asked Questions
Associated Services
We leverage template-independent enzymatic methods to produce long, complex DNA sequences with high fidelity, ideal for applications demanding precision in gene synthesis or repetitive regions. To further optimize these sequences for functional performance, we offer Oligonucleotide-based Base Modification Services, which fine-tune stability, binding affinity, and therapeutic efficacy:
At CD BioGlyco, we are experts in DNA synthesis and the Synthesis of Modified DNA Molecules with years of experience. With our state-of-the-art technology, our exceptional research team is fully prepared to meet your specific requirements. We devote ourselves to delivering high-quality custom DNA synthesis solutions. We appreciate you to contact us, as we are excited to have the chance to support you.
Reference
- Forget, S.M.; et al. Evolving a terminal deoxynucleotidyl transferase for commercial enzymatic DNA synthesis. Nucleic Acids Research. 2025, 53(4): gkaf115. (Open Access)
