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From DNA To Reality: Understanding the Process of Gene Synthesis

Dec 1st 2023

From DNA To Reality: Understanding the Process of Gene Synthesis

Gene synthesis technology has revolutionized fields like biotechnology and synthetic biology by enabling researchers to construct genetic material in the lab artificially. This process of synthesizing designed gene sequences from scratch has unlocked innovative applications across healthcare, agriculture, biofuels, and more.

With gene synthesis, researchers gain the power to harness biology in new ways that are not possible with natural gene cloning alone. By synthesizing genes to specification, scientists can engineer novel biochemical pathways, create protein variants with new properties, program genetic behaviors, correct defective disease-causing genes, and more.

This article will explore the essential steps in converting digital DNA sequence information into physical, synthetic genes using current laboratory gene synthesis methods and technologies. We will cover key aspects of the process, including gene design optimization, high-throughput oligonucleotide synthesis, gene assembly protocols, sequence validation, and the impact of synthetic genetics.

gene synthesis process

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Overview of Gene Synthesis

Gene synthesis involves chemically constructing artificial genetic material in the lab without isolating DNA from an existing organism. To start, researchers design their target gene sequence digitally using a computer. The next step is physically building the gene base-by-base from raw nucleotide building blocks to match the digital blueprint. The gene synthesis process typically relies on software algorithms to optimize the design, high-throughput oligonucleotide synthesis to construct short fragments, and molecular biology techniques to assemble the oligos into the final gene. This workflow converts the digital gene sequence into lab-fabricated DNA ready for experiments.

Early techniques for chemical DNA synthesis were developed in the 1970s, but significant advances in gene synthesis technologies have now made the process routine, affordable, and accessible. Gene synthesis services empower ordering genes to specification for various applications.

Gene Design Software

The first critical step in gene synthesis is designing the optimal gene sequence using specialized software algorithms. Researchers input the target protein's amino acid sequence into the software. The program then back-translates this protein sequence into DNA codon sequences that encode it. Additional gene design optimization is performed to ensure the final sequence has the best chance for effective synthesis and functioning when complete.

Proper gene design lays the foundation for the rest of the gene synthesis workflow. Elements like codon optimization, minimizing repetitive regions, removing toxic components, and incorporating cloning features enable the resulting gene to be accurately synthesized and properly expressed in the host organism. Careful gene design considering these parameters is essential for the success of the overall process.

Key Gene Design Features: Crafting the Blueprint for Successful Synthesis

Designing a synthetic gene involves strategic decisions to ensure optimal expression, functionality, and compatibility with the chosen host organism. Each design element contributes to the overall success of gene synthesis, culminating in a functional gene that behaves as intended when expressed in cells. Here, we delve into the vital gene design features that shape the blueprint for successful synthesis.

DNA synthesis, gene synthesis methods

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1. Codon Optimization for Expression Organism

A pivotal facet of genetic blueprinting lies in codon refinement, the endeavor to alter a gene'scodon utilization to harmonize with the requisites of the host entity. Given the divergent utilization of codons across different organisms, augmenting codon adeptness amplifies translational effectiveness. Selecting an expression framework that guarantees the synthetic gene's translation aligns seamlessly with the targeted protein elevates manifestation proficiency and biological potency.

2. Minimizing Repetitive Sequences

Redundant patterns can impede the fabrication and manifestation of a gene. Such patterns might induce misfolding, compromised structural stability, or complications in gene replication. It is prudent to minimize the presence of repetitive sequences during gene design to uphold the stability of the synthesized gene and avert unwarranted structural challenges. This attribute contributes to enhancing the dependability and efficiency of subsequent processes, such as cloning and expression.

3. Removing Toxic Elements like Restriction Sites

Restriction sites represent genetic components with the capacity to jeopardize the well-being of the host organism or compromise the intended functionality of the artificial gene. These elements undergo meticulous exclusion in the gene crafting phase to avert adverse impacts on the host. Besides fortifying the health of the expression organism and expediting the ensuing stages of gene synthesis, this practice facilitates adept gene cloning and expression.

4. Adding Cloning Features and Fusion Tags

Integration of fusion tags and cloning attributes is integral to the construction of synthetic gene designs aimed at simplifying the processes of cloning and protein purification. The engineered gene seamlessly integrates into expression vectors, courtesy of cloning features, encompassing recognition sites for restriction enzymes.

Conversely, the inclusion of fusion tags streamlines the purification of the resultant protein. These design components augment the adaptability and applicability of the synthetic gene, rendering it suitable for diverse experimental setups and purposes.

5. Defining Oligo Synthesis Fragmentation

Oligo synthesis fragmentation is a critical step in the gene synthesis process. The software used in gene design breaks down the complete gene sequence into smaller fragments, typically ranging from 150 to 200 base pairs (bp). This fragmentation serves multiple purposes. Firstly, it facilitates the synthesis of each oligo, making the overall gene synthesis process more manageable. Secondly, smaller fragments improve synthesis accuracy, minimizing errors and ensuring the fidelity of the final gene sequence.

Elaborating on Oligonucleotide Synthesis Phases: A Molecular Symphony

Oligonucleotide synthesis, the delicate art of crafting short DNA fragments, unfolds in two distinctive phases—solid and liquid phases. Each phase orchestrates a symphony of chemical reactions, transforming nucleotides into the molecular building blocks essential for gene assembly.

Solid Phase Synthesis: Anchored Precision

Solid phase synthesis is akin to constructing a molecular tower, where nucleotides are sequentially added to a growing chain anchored to a solid resin. This solid support, often a resin bead, acts as an immovable anchor for the synthesis process. The synthesis initiates by attaching the first nucleotide to the solid support, typically through its 3' end. Subsequent nucleotides are then added, one at a time, to the 3' end of the growing chain.

Liquid Phase Synthesis: Crafting in Solution

In liquid phase synthesis, the process begins by pre-assembling the oligonucleotide fragments in solution. Though initially in solution, these fragments are strategically designed with specific sequences. After pre-assembly, they are immobilized onto a solid support for extension.

Choosing the Right Phase: Balancing Precision and Flexibility

The selection between solid-phase and liquid-phase production depends on the particular needs of the production project. With its step-by-step accuracy and regulated setting, solid-phase production is frequently favored for regular oligonucleotide production duties. In contrast, liquid-phase production provides more adaptability in planning and solution chemistry, making it beneficial for projects with particular customization requirements.

Gene Assembly

Building an entire gene from separate single-stranded oligos is an intricate job that includes various specific molecular biology methods. These methods smoothly interlace the produced oligonucleotide pieces, forming a functional and precise depiction of the desired gene. Let's explore the specifics of these molecular instruments:

Synthetic gene production

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1. Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) is a cornerstone technique for gene construction. It involves repeated cycles of oligo extension and amplification using a DNA polymerase enzyme. Each process consists of denaturation, primer annealing, and extension. The denaturation step separates the DNA strands, while primers, short oligos designed to match the ends of the target gene, anneal to the single-stranded DNA. DNA polymerase then extends the primers, creating a new complementary strand. After multiple cycles, the target gene is amplified exponentially.

2. Ligation

Ligation includes the enzymatic linking of oligos end-to-end using DNA ligase. Each oligo will overlap with its nearby partner, forming a smooth connection. This method is advantageous when forming lengthier genes or when PCR amplification is problematic.

3. Recombination

Recombination uses host organisms' in vivo DNA repair systems to assemble DNA fragments. In synthetic biology, homologous recombination is frequently used. The host organism identifies overlapping regions in the oligos and naturally includes them in the genome through DNA repair mechanisms.

4. Hybridization

Hybridization involves the annealing of complementary oligos into larger fragments. Each oligo is designed to pair specifically with its complementary counterpart. The resulting hybridized fragments can then be further extended or ligated to form longer sequences.

Construct Verification

Following gene synthesis and assembly, the accuracy of the construct is verified:

  • Sequencing: Ensures the sequence matches the original design
  • Restriction mapping: Confirms gene can be cut at expected sites
  • Cloning: Demonstrates gene stability when propagated in cells
  • Functional testing: Validates gene expresses designed protein product

Applications of Synthetic Genes

Researchers are leveraging custom gene synthesis for innovative applications, including:

  • Metabolic engineering: Design of novel biochemical pathways
  • Protein engineering: Constructing variants with altered properties
  • Synthetic biology: Programming genetic circuits and behaviors
  • Gene therapy: Correcting defective genes underlying disease
  • Drug screening: Producing gene targets to enable compound assays
  • Vaccine development: Design of optimized antigen genes
  • Agricultural biotech: Improving crops via transgenesis

The ability to synthesize any gene sequence allows bioengineers to test concepts impossible with natural gene cloning alone. Continued advances in gene synthesis technologies will enable previously unthinkable manipulations of biology.

The Critical Role of Gene Synthesis Companies

The advent of gene synthesis technologies has enabled the rise of dedicated biotech companies focused on providing gene synthesis services. By developing expertise and infrastructure for high-throughput gene construction, these companies have transitioned custom gene synthesis from specialized academic labs into an accessible on-demand service.

Researchers across industrial and academic settings now rely on gene synthesis companies to provide ready access to synthetic gene capabilities without building internal gene synthesis competence.

Benefits of Gene Synthesis Services

Key benefits offered by gene synthesis service providers include:

  • Sophisticated gene design software incorporating decades of optimization algorithms. This automates sequence processing from protein to DNA codon optimization.
  • They establish pipelines for massively parallel oligonucleotide synthesis - Companies maintain large synthesis arrays to construct thousands of oligos in parallel.
  • Extensive experience synthesizing complex long genes, viral genomes, gene clusters, and DNA libraries to specification. Difficult sequences are routine for dedicated providers.
  • Range of advanced assembly methods like Gibson, Golden Gate, and more. Providers offer multiple techniques to cover diverse construct needs.
  • Rigorous quality control using NGS, digital PCR, and functional assays. This ensures the accuracy and integrity of synthesized sequences.
  • Rapid turnaround times from order to gene delivery - Large synthesis capacity enables fast delivery even for large custom orders.
  • Bioinformatics, cloning, cell line development, and other services to support projects from design to experimentation. Fully integrated workflows prevent gaps.

By providing on-demand access to industry-leading gene synthesis capabilities, companies empower academic and industrial labs to take on ambitious synthetic biology and genetic engineering projects that would otherwise be infeasible due to gene synthesis bottlenecks. This democratization promises to accelerate both discovery research and biotechnology innovation.

Key Partnerships Driving Innovation in Gene Synthesis

Several key partnerships among industry participants have been crucial to propelling innovation and advancement in gene synthesis technologies:

1. ELISA Kit Manufacturers - Collaboration Essential for Diagnostics

ELISA kit manufacturers play an integral role in diagnostic testing applications. The development of these kits relies heavily on synthesized genes and proteins provided by gene and protein synthesis companies. This highlights the importance of integrating molecular biology and diagnostic technologies through strategic partnerships.

2. Protein Synthesis Companies - Revolutionizing Biomanufacturing

Protein synthesis companies enable industrial scale biomanufacturing of recombinant proteins. By offering integrated services spanning DNA synthesis, protein expression, and purification, they provide a streamlined solution for producing large quantities of biomolecules. The convergence of gene and protein synthesis services has transformed the bioproduction landscape.

3. Peptide Synthesis Companies - Tailoring Molecules with Precision

Peptide synthesis companies are vital for engineering customized functional peptides. Partnerships with gene synthesis providers allow tailored peptides to be seamlessly integrated into synthetic gene designs, enhancing the precision of molecular engineering.

4. Antibody Production Companies - Driving Therapeutic Innovation

Providers of custom antibody services are advancing therapeutic antibody development. Comprehensive solutions for novel therapeutics are unlocked by combining gene synthesis with antibody discovery platforms.

The Importance of Peptide Synthesis Economics

In addition to these key partnerships, examining peptide synthesis costs and providers gives an important economic context. As researchers search for affordable solutions, understanding price dynamics for custom peptide synthesis services is essential.