Sequencing Reaction Clean-up
Although every single step in the Sanger sequencing workflow plays an important role in generating good sequencing results, two major criteria are critical for high-quality results from Sanger sequencing: high template quality and quantity and thorough clean-up of the sequencing reactions. Variations in these criteria may contribute to uncertainty in sequencing success.
Clean-up of sequencing reactions is performed to remove unincorporated dyes (fluorescent labeled ddNTPs), dNTPs, primers, and enzymes from the completed reaction mixture. Presence of these contaminants can generate poor quality of the sequence readings.
Cleaning methods for cycling sequencing reactions
Different methods can be used for clean-up of the cycling sequencing reactions, including size exclusion, affinity purification, ethanol precipitation, binding with magnetic beads (such as ADS™ Sequencing Reaction Cleaning Beads) and proprietary methods (such as in ADS™ BD XT purification kit). Although ethanol precipitation is the most cost-effective method, sequencing quality obtained is often poorer the first 40 nucleotides close to the primer compared to above methods.
Sequencing reaction sample purification with specialized resins or magnetic beads
We developed two clean-up products, ADS™ BD-XT Purification Kit and ADS™ Sequencing Reaction Cleaning Beads for different clean-up preferences. The ADS™ BD-XT kit uses specialized resin for contaminant binding and can reduce sequencing signal loss and prevent any potential contamination during the wash and elution steps, without repeating washing steps.Therefore, the sequencing signal strength is often better with the ADS™ BD-XT kit compared to magnetic beads. However, use of magnetic beads is cost-effective and is best for large-scale sequencing to save sequencing cost while still achieving high-quality sequencing results.