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Sequencing Product Cleanup

Sequencing Product Cleanup

Removal of unincorporated dye terminators and impurities from cycle sequencing reactions is one of the major bottlenecks in the sequencing workflow before loading samples onto a sequencer.

Methods such as ethanol precipitation, sephadex filtration, and other non-magnetic bead based systems, require steps that are not well suited for full automation. The D-Pure™ DyeTerminator Removal Kit from NimaGen is ideally suited to remove salts and unincorporated dyes in a fully automated procedure with the Microlab® STARlet.

This application greatly increases throughput and reproducibility in sequencing sample purification.

Sanger sequencing is still the gold standard for genetic variation analysis. Most DNA sequencing laboratories require highly automated, fast, robust and highly standardized workflows, without compromises in data quality. This resulted in the widespread adoption of robotic liquid handlers in processes like linear amplification (cycle sequencing) using PCR products as a template, cleanup and re-suspension in a buffer solution for sequencing. One of the major bottlenecks in the sequencing workflow is removal of unincorporated dye terminators and salt ions from the sequencing reactions before loading onto a sequencing device.

Methods such as ethanol precipitation, sephadex filtration, and other, non-magnetic bead based systems, require manual steps such as centrifugation and/or vortexing and are therefore not well suited for full automation. In this application we describe how removal of salts and unincorporated dyes using D-Pure™ DyeTerminator Removal Kit from NimaGen, www.nimagen.com, in a fully automated procedure with the Microlab® STARlet greatly increases throughput and reproducibility in sequencing sample purification.

Features and Benefits

  • Co-developed between Hamilton, Radboud University Medical Center and NimaGen
  • Fully automated sequencing reaction purification with maximum throughput, quality and reliability.
  • Simplified workflow: no need for manual intervention such as centrifugation or vortexing
  • High purity: samples are clean and ready for analysis on an automated sequencer
  • Reduced sample-to-sample variability: high sample stability, low peak degradation, very low variation in signal strength, reduction of overloaded samples, less need for re-injection, less effort for signal normalization, compared to manual purification.

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Removal of unincorporated dye terminator after DNA sequencing reactions is a prerequisite to loading samples on automated capillary sequencers. Agencourt CleanSEQ® uses Solid Phase Reversible Immobilization (SPRI™) of DNA products on magnetic microparticles. It is a rapid and effective method for removal of unincorporated dye terminator. The chemistry has been automated on the HAMILTON MICROLAB® STAR and requires minimal deck components. The flexibility of the STAR and the robustness of the method facilitate implementation in low or high throughput environments. The method can be used with washable needles or an economical quantity of disposable tips. The application was optimised with regard to minimal costs for reagents and consumables, high quality data and maximum read length.

Features and Benefits

  • Low running costs due to use of washable steel needles for complete method
  • Throughput can easily be increased due to scaleability of the method and flexibility of the system
  • Fully automated hands-free processing with built-in robotic plate-handler (iSWAP or CO-RE grip)
  • Reproducible high quality data with extended read lengths and absence of dye blobs
  • Other applications like PCR reaction cleanup with Agencourt kits can easily be automated on the same platform

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