About Dye Terminator Cycle Sequencing Clean-up

Clean-up is a critical step in a sequencing workflow, preparing DNA or RNA samples to be compatible with a sequencer. Sanger Sequencing, also known as Capillary Electrophoresis (CE) sequencing, is a method of DNA sequencing, based on the selective incorporation of fluorescent dideoxynucleotide chain terminators (dye terminators) by DNA polymerase during in vitro DNA replication (cycle sequencing). As sequencing is a highly accurate technique based on reading a DNA sequence base by base, it is very important to have an effective clean-up of dye-labeled extension products in the reaction mixtures, eliminating interference of unincorporated dye terminators, desoxynucleotides and salt-ions after Sanger cycle sequencing. This prevents their co-injection with sequencing products during Capillary Electrophoresis.

Clean-up of PCR samples prior to sequencing and removal of dye terminators post cycle sequencing can be performed using magnetic bead, resin-based, and enzymatic methods. In most instances, a magnetic bead-based chemistry is utilized to generate capillary electrophoresis-ready and sequence-ready samples.

Magnetic beads selectively bind to the Sanger cycle sequencing extension products. Products bound to the magnet are then pulled to one side, so all artifacts and contaminants can be effectively washed away. Subsequent wash steps complete the clean-up of cycle sequencing products, followed by elution of the extension products from the beads into solution. Purified dye-labeled extension products will be directly loaded on Capillary Electrophoresis (Genetic Analyzer) without the need for resuspension.

Magnetic clean-up protocols can be performed directly in the thermal cycling plate, and can be processed both manually and automated. A key advantages of using magnetic beads in clean-up is that the large surface area of the beads allows for highly efficient binding of nucleic acids, meaning better washing efficiency when compared with resin-based or enzymatic methods. The lack of centrifugation steps that can produce shear forces and cause breaking of nucleic acids is thought to better maintain intact longer DNA fragments.

Typically, resin is added to the finished Sanger cycle sequencing products and vortexed, allowing the resin to capture and immobilize unincorporated dye terminators and salt-ions. The captured components are then moved to the bottom of the reaction vessel by brief centrifugation. The purified dye-labeled extension products remaining in the supernatant are then injected directly from the supernatant into the Genetic Analyzer.

Resin-based procedures optimized for use in conjunction with dye terminator cycle sequencing do not require repeated wash steps, minimizing sample loss from either long or short fragments of extension products.

Fluorescent or radioactive DNA sequencing, Single Nucleotide Polymorphism (SNP) analysis, genotyping, and many other applications require PCR products which are free of excess desoxynucleotides and primers, that can lead to high background and base miscalling.

Novel enzymatic clean-up methods, like ExoSAP-IT™ and its equivalent ExS-Pure™, utilize two hydrolytic enzymes, Exonuclease I and Shrimp Alkaline Phosphatase, together in a specially formulated buffer. Exonuclease I removes residual single-stranded primers and any extraneous single-stranded DNA produced in the PCR. Shrimp Alkaline Phosphatase removes the free desoxynucleotides from the PCR mixture which would interfere with the subsequent (cycle sequencing) reaction.

ExoSAP-IT™ and ExS-Pure™ are recommended for PCR product clean-up prior to dye terminator cycle sequencing. Enzymatic clean-up reagent is added directly to the PCR product and incubated at 37°C for 15 minutes. These enzymes are active in the buffer used for PCR, so no buffer exchange is required. After PCR treatment, the enzymes are inactivated simply by heating between 80 to 90 °C for 15 minutes.

ExoSAP-IT is a trademark of Affimetrix. ExS-Pure is a trademark of NimaGen.