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Primer Walking – Sequencing of Long DNA Templates

Primer Walking
 
 
Complete sequencing of plasmids and long DNA fragments without manual primer design. Microsynth designs primers, performs sequencing and assembles your full sequence. Ideal for DNA templates longer than 1,400 bp and complex sequencing projects.
 

 

Why Choose Primer Walking?

  • Complete sequencing solution – no need to design primers or plan sequencing strategy
  • Sequencing of long DNA templates – ideal for plasmids and large PCR fragments
  • Fast progress – up to ~1,600 bp per day
  • High accuracy – double-stranded sequencing up to 99.99% per base
  • Automated primer design & synthesis – performed in-house

 

Applications

  • Confirmation of sequence integrity for plasmids and PCR products
  • Full-length sequencing of long DNA templates (>1.4 kb)
  • Troubleshooting of failed amplifications or unexpected restriction digest patterns
  • Sequencing of complex or difficult regions (e.g. GC-rich sequences or repeats)
  • Full sequencing of AAV plasmids and challenging ITR regions

 

What is Primer Walking?

Primer Walking is a Sanger sequencing strategy used to determine the full sequence of long DNA templates.

Instead of sequencing a fragment in a single reaction, new sequencing primers are designed step by step based on previously obtained sequence data. This allows continuous sequencing across the entire DNA molecule.

Microsynth performs the complete process, including primer design, synthesis, sequencing and sequence assembly.

 

How It Works

  1. Submit your DNA sample and (optionally) a reference sequence
  2. Microsynth designs sequencing primers step by step
  3. Sequential sequencing reactions are performed
  4. Sequences are assembled into a full-length contig
  5. Optional verification against your reference sequence

 

Output & Data

You receive:

  • Chromatogram files for each sequencing reaction
  • Assembled sequence (contig)
  • Project data sheet including sequencing strategy
  • Optional verification against reference sequence

Accuracy & Quality

  • Single-stranded or double-stranded sequencing options
  • Double-stranded sequencing ensures accuracy of ≥99.99% per base
  • Continuous quality control during sequence assembly

 

How to Order

Procedure to use our Primer Walking Service for sequencing:
  • Enter our webshop
  • Click on Tube Standard Service under "Primer Walking" and follow the further instructions