Stepped Elongation Time protocol (STeP) (1) is an optimized method of sequence cycling used for Sanger sequencing. The STeP protocol aimed to increase the efficiency of standard DNA sequencing by reducing the amount of time it takes to perform sequencing. However, the original protocol described by Platt et al., published in 2007 in the journal BioTechniques, lacks critical experimental details and no revised version has been published to date. Interestingly, this original paper continues to remain highly cited with 35% of its total citations in the past four years, a metric that suggests it is still receiving higher than expected interest according to Dimensions (2), the world’s largest linked research information dataset. We present here an updated Stepped Time Elongation protocol (STeP-Up), which aims to improve on the useability and showcase cost effectiveness and DNA sequencing efficiency, while maintaining the highest quality results for standard DNA sequencing.
We have validated this protocol for both PCR amplicon and plasmid DNA sequencing, correlated specified ranges of template DNA and confirmed that significantly less reagents are required than that suggested by standard sequencing protocols. Experimental details that were missing from the original STeP protocol that we have clarified in STeP-Up include recommended DNA quantities base on template type, appropriate reaction component set-up with concentrations and volumes and clearly defined cycling parameters. The standard BigDye cycle sequencing reaction (3) takes approximately 2.5 hours to run on a thermocycler, whereas the STeP protocol takes 55 minutes to complete. One caveat to Sanger sequencing is the expense associated with the reagents. For example, one of the core reagents, BigDye Terminator v3.1 Ready Reaction Mix, has remained expensive over a decade after STeP was developed. Therefore, by decreasing the amount of reagent used in the BigDye Fast and standard sequencing protocols, the experimental costs can be greatly reduced, and more reactions can be performed, increasing overall sequencing efficacy.