Services

Professional and Cost-Saving Solutions

  1. Home
  2. Enzyme Engineering and Modification
  3. Enzyme Engineering
  4. Enzyme Engineering by Site-directed Mutagenesis
  5. Upstream Services for Site-directed Mutagenesis
  6. Template DNA Sequencing for Site-Directed Mutagenesis

Template DNA Sequencing for Site-Directed Mutagenesis

inquiry

Template DNA sequencing is a critical upstream step in enzyme engineering by site-directed mutagenesis (SDM). Accurate sequencing of the parental (template) DNA ensures that mutations are introduced into a verified and error-free genetic background, eliminating the risk of false-positive results or downstream expression failures. At Creative Enzymes, we provide high-fidelity sequencing and verification services designed to confirm the integrity, identity, and readiness of template DNA prior to mutagenesis. This service guarantees precise mutation design, reliable construct generation, and smooth transition into cloning and expression stages.

Template DNA Sequencing: The Foundation of SDM Workflow

Site-directed mutagenesis is a powerful molecular tool for enzyme engineering, enabling precise modification of amino acid residues to enhance catalytic activity, stability, or substrate specificity. However, the success of SDM fundamentally depends on the quality of the starting DNA template. Even minor sequence discrepancies or undetected polymorphisms can lead to unintended mutations, compromised enzyme function, or experimental inconsistencies.

Therefore, template DNA sequencing serves as the foundation of a robust SDM workflow. By validating the entire gene sequence and its vector context before mutation introduction, researchers can ensure experimental accuracy and reproducibility. Creative Enzymes integrates advanced sequencing technologies and stringent bioinformatic validation to confirm that every template used for mutagenesis meets the highest quality standards.

A simplified workflow of a site-directed mutagenesis strategy, starting with template DNA sequencingFigure 1. A site-directed mutagenesis strategy. The first step is to prepare the template DNA. (Allemandou et al., 2003)

Template DNA Sequencing Services

Creative Enzymes delivers a complete sequencing and validation solution tailored to mutagenesis-based enzyme engineering projects. Our service includes:

  • Comprehensive template DNA verification, covering full-length gene sequencing and vector backbone confirmation.
  • Error-free sequence validation using dual-strand and high-coverage reads.
  • Sequence alignment and annotation against reference or design sequences.
  • Quality assessment and report generation, including chromatogram analysis and confidence scoring.
  • Optional plasmid purification and QC prior to downstream mutagenesis.
  • Customized sequencing strategy (Sanger or NGS) according to template size and complexity.

Service Workflow

Service workflow of template DNA sequencing for site-directed mutagenesis

Service Details

Parameter Specification
Sample Types Accepted Plasmid DNA, PCR products, synthetic genes
Recommended Concentration ≥100 ng/µL (plasmid); ≥50 ng/µL (PCR product)
Purity Requirement A260/A280 ratio of 1.8–2.0
Turnaround Time Typically 3–5 business days
Deliverables
  • Verified DNA sequence and chromatograms
  • Quality assessment summary (coverage, accuracy, base-calling confidence)
  • Sequence alignment and mutation-readiness certificate
Optional Add-ons
  • Gene synthesis from verified sequence
  • Subcloning into expression vectors
  • Storage and documentation of verified templates for future use

Related Services

Our Template DNA Sequencing service forms one of the three pillars of Upstream Services for Site-Directed Mutagenesis, providing a seamless link between gene design, verification, and vector preparation. Explore our related services to complete your project workflow:

Together, these services ensure a consistent, validated, and high-quality foundation for successful enzyme engineering by site-directed mutagenesis.

Inquiry

What Sets Us Apart

High-Accuracy Sequencing

Dual-platform sequencing (Sanger + NGS) ensures base-level fidelity and confidence.

Expert Bioinformatics

In-depth sequence validation and comparative alignment by experienced molecular biologists.

Seamless Integration

Fully compatible with our site-directed mutagenesis and expression services, streamlining project continuity.

Customized Strategies

Flexible primer design, sequencing coverage, and analytical depth tailored to project needs.

Fast Turnaround and Reliable Delivery

Efficient logistics and project management minimize waiting time.

Comprehensive Data Reporting

Clear, publication-ready sequencing reports with full traceability and certification.

Template DNA Sequencing for SDM: Case Studies

Case 1: Template Verification Preventing Downstream Mutation Errors

Client Need:

A research team preparing to introduce multiple point mutations into a hydrolase gene planned a series of structure–function studies. Before initiating site-directed mutagenesis (SDM), they requested sequence verification of their plasmid template to ensure experimental accuracy.

Our Approach:

We performed full-plasmid Sanger sequencing using overlapping primer sets to ensure complete coverage of the coding region and adjacent regulatory elements. Our analysis uncovered two unexpected silent polymorphisms and, more critically, a single-base deletion within the open reading frame—an error that would have shifted the reading frame and compromised all planned mutations. After reporting the discrepancies, we re-synthesized and re-cloned the correct gene sequence, providing the client with a fully validated template ready for SDM.

Outcome:

Using the corrected construct, the client successfully generated all intended mutants, each exhibiting proper folding and expected hydrolase activity.

Case 2: High-Throughput Template Screening for Parallel Mutagenesis

Client Need:

A biotechnology partner working on thermostable oxidoreductases sought to perform parallel mutagenesis across a panel of 40 enzyme variants. Because the constructs originated from different cloning batches, the team required rapid and reliable sequence validation prior to initiating high-throughput engineering and expression trials.

Our Approach:

We implemented a high-throughput sequencing workflow using a 96-well plate format, enabling simultaneous processing of all 40 plasmids. Automated template preparation, streamlined primer design, and multiplexed Sanger sequencing allowed full coding sequence verification within five days. Each sequence was analyzed for point mutations, frameshifts, truncations, and unintended cloning artifacts. Verified constructs were compiled into a quality-checked template library ready for downstream mutagenesis.

Outcome:

The validated template set enabled the client to proceed directly with parallel mutagenesis and expression screening. Subsequent assays identified multiple oxidoreductase variants with significantly enhanced thermostability.

Template DNA Sequencing for SDM: FAQs

  • Q: Why is template DNA sequencing necessary before site-directed mutagenesis?

    A: Sequencing ensures that the parental DNA accurately represents the intended wild-type sequence. Any errors or polymorphisms in the template can result in failed mutagenesis or incorrect protein expression.

  • Q: What sequencing methods do you use?

    A: We employ both Sanger sequencing for short to mid-length genes and Next-Generation Sequencing (NGS) for complex or long templates, ensuring full coverage and precision.

  • Q: How much DNA should I submit?

    A: Typically, 10–20 µg of high-purity plasmid DNA or 200–500 ng of PCR product is sufficient for sequencing and quality control.

  • Q: Can you sequence GC-rich or repetitive regions?

    A: Yes. We use optimized chemistry and primer design strategies to overcome difficult regions, ensuring reliable reads.

  • Q: Will you keep a record of my verified sequence?

    A: Yes. With client consent, we securely store validated sequences and documentation to facilitate future mutagenesis or expression projects.

  • Q: Can this service be combined with your mutagenesis workflow?

    A: Absolutely. Template sequencing is fully integrated with our upstream services for site-directed mutagenesis, ensuring verified constructs are immediately ready for mutation introduction and downstream applications.

References:

  1. Allemandou F, Nussberger J, Brunner HR, Brakch N. Rapid site‐directed mutagenesis using two‐PCR‐generated DNA fragments reproducing the plasmid template. BioMed Research International. 2003;2003(3):202-207. doi:10.1155/S1110724303209141
Related Services
Online Inquiry

For research and industrial use only. Not intended for personal medicinal use. Certain food-grade products are suitable for formulation development in food and related applications.

Services
Online Inquiry

For research and industrial use only. Not intended for personal medicinal use. Certain food-grade products are suitable for formulation development in food and related applications.