Importance of cleaning cycle sequencing reactions

In the sanger sequencing workflow, cycle sequencing reaction is certainly the core and critical step because the reaction determines if the extension products are appropriately synthesized and labeled with great fluorescent signal intensity. However, the quality of the sequencing reactions is dependent on the quality and quantity of the template and primer used. In addition, to maximize the quality of Sanger sequencing, the cycle sequencing reactions need to be purified to remove the extra salts, and unincorporated dye-labeled precursors (ddNTPs) left in the reaction, both of which could interfere with downstream capillary electrophoresis (fragment migration) and data analysis (peak reading). For instance, the left-over unincorporated ddNTP terminators form high-intensity signals (dye blobs) in the electropherograms blocking up to 20 bases around 70 base pair sequences.

Methods for purification of cycle sequencing reactions

Different commercial kits are available to purify the cycle sequencing reactions. They are based on either size-exclusion, affinity, magnetic beads, or special resin. Compared to the most cost-effective ethanol precipitation method, these products generally provide the benefit of less hands-on operation, less sample loss, and generally cleaner background. While I would like to hear from you about your favorite products, I want to take the opportunity to introduce what AdvancedSeq offers.

Sequencing cleaning reaction kit and SupreDye XT purification kit

AdvancedSeq has ADS sequencing cleaning beads and SupreDye XT purification kit that are independently used for cleaning of cycle sequencing reactions. These two products use different purification mechanisms and users can choose one of them based on their preference.

The ADS sequencing cleaning beads product is more cost-effective and optimized for cleaning with minimum washing steps. The cleaning beads, which is a type of magnetic beads, clean the sequencing reactions in a 96-well format with multi-channel pipettes. The fluorescence-labeled extension products bind to the magnetic beads while other components are left in the solution. After one round of brief wash, the purified extension products are eluted in water or low ionic TE buffer. You can download our app note for detailed performance comparison of our sequencing cleaning beads with other products.

As an alternative to BigDye XTerminator purification kit, SupreDye XT purification kit, on the other hand, uses special resin to bind the components such as unincorporated dyes (ddNTPs) whereas the labeled extension products are left in solution. Without any wash steps, the resin binding to the unwanted reaction components through vortex is spun down and the extensive products in the solution can be directly loaded for capillary electrophoresis.

If you want to provide your thoughts on how you choose a purification product, please leave a comment below. We will be happy to hear from you.