Primer Walking


Primer Walking utilizes multiple primers in a series to sequence entire plasmids or PCR products. GENEWIZ Primer Walking service options include plasmid sequence confirmation for already known sequences and plasmid sequence discovery for unknown sequences. For AAV vectors, we offer primer walking plus our new, proprietary AAV-ITR Sequencing. Trust GENEWIZ from Azenta to manage your project from primer design to variance analysis and report generation.



Azenta’s primer walking sequence confirmation service offers peace of mind and helps you perform plasmid identity verification thus avoiding costly complications with downstream applications. Estimated turnaround time is 3-5 business days after primer design. We also offer GLP level plasmid confirmatory sequencing.

With our plasmid confirmation service you can:

• Confirm the sequence of your cloned products/genes of interest
• Sequence your entire plasmid, including difficult-to-amplify regions
• Examine failed amplifications and incorrect restriction digest patterns
Sequence full length AAV plasmids and hard to sequence ITR regions


1. Submit Samples

2. Primer Design*

3. Sanger Sequencing

4. Consensus Sequence Assembly

5. Data Report

*Please note, a reference sequence is required for primer design.


Azenta’s primer walking sequence discovery service maps your unknown plasmid or PCR product for use in downstream applications. Subsequent primers will be designed as sequences are generated. Estimated turnaround time is 3-5 days per 1kb.


With our plasmid discovery service you can:

• Create a plasmid map for your undetermined plasmids
• Confirm the sequence of your mystery constructs
• Identify unknown regions of your plasmids
Sequence full length AAV plasmids and hard to sequence ITR regions

*As a starting point, an antibiotic resistance gene, a known promoter (M13, T7), or another known sequence will be required for the initial primer design.


Azenta has developed a proprietary Sanger sequencing method specifically designed to sequence the inverted terminal repeat (ITR) hairpin regions of adeno-associated virus (AAV) plasmids. Our proprietary protocol prevents the abrupt reduction in sequencing signal at the start of the ITR hairpin and reads through the full length of the ITR region.

AAV-ITR sequencing can be incorporated into either our sequence discovery or sequence confirmation services.


ITR sequencing using standard protocol


ITR Sequencing using GENEWIZ's proprietary protocol


Primer Walking Features & Benefits

Industry-leading turnaround time starting at 3-5 business days for sequence confirmation
Single-stranded, double-stranded, or 4x coverage available
Can be combined with our ITR Sanger sequencing service for complete sequencing of AAV plasmids
Any size plasmid prep or PCR of difficult templates are available upon request
Dedicated Ph.D.-level support at every step with real-time project updates through our online system
Contact us to locate a drop box or pick-up service near you for free shipping

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AAV ITR Sequencing Case Study

Tech Note: Reading Through the Inverted Terminal Repeats (ITRs) of Adeno-Associated Virus (AAV)


Azenta has developed a high-quality, direct Sanger sequencing method that reads through both intact and commonly mutated inverted terminal repeat (ITR) regions of adeno-associated virus (AAV). This method is an effective tool for assessing the integrity of ITRs in AAV plasmids.


*Samples must arrive at the GENEWIZ New Jersey Laboratory before 10:00 am EST to qualify for Same Day service. Note that direct-sequencing templates are not available for our Same Day service.

Email | Phone (1-877-436-3949, Ext. 2)