The SequiTherm EXCEL™ II DNA Sequencing Kit: A New Kit for Both Cycle and Non-Cycle Sequencing

The original SequiTherm EXCEL™ Kits, developed for cycle sequencing, generated data from many difficult DNA templates that were not resolved using other cycle sequencing kits.1,2 SequiTherm EXCEL II DNA Polymerase was then incorporated into the SequiTherm EXCEL Kits. This new polymerase produced greater signal intensities, enabling sequencing of up to five times less template than the original kit and reducing autoradiographic exposure times. It also allowed sequencing of double-stranded (ds) DNA templates using a high-temperature isothermal (non-cycle) protocol with labeled primers, using only thermal denaturation to gain accessibility of the primer to the primer binding site.3 In this article, we introduce the new SequiTherm EXCEL II DNA Sequencing Kit. The SequiTherm EXCEL II Kit offers users the broadest choice of sequencing methods available in a single kit. These methods include cycle sequencing and high-temperature isothermal sequencing with either labeled primers or internal label incorporation using 32P, 33P, or 35S radionuclides. The SequiTherm EXCEL II DNA Sequencing Kit retains all the advantages of the previous kits and has proven advantages over other cycle and non-cycle sequencing kits.

In the following experiments, we demonstrate the capabilities of the SequiTherm EXCEL II DNA Sequencing Kit in non-cycle sequencing reactions using [alpha-35S]-dATP label. We show the speed and flexibility of the SequiTherm EXCEL II protocol and the use of low amounts of 35S label in a reaction. We demonstrate the ability to sequence difficult templates, generate data close to the primer, and produce extended sequence reads using the SequiTherm EXCEL II Kit. In addition, we compare the SequiTherm EXCEL II Kit with a standard cycle sequencing kit in 35S-internal-label and 32P-end-label cycle sequencing reactions.

Methods and Discussion

Protocol speed and flexibility

Time requirements, flexibility, and simplicity of protocol are important factors with respect to kit convenience. The three main steps required in the non-cycle sequencing of dsDNA are template denaturation, primer annealing, and primer extension/termination. Table 1 shows a comparison of the SequiTherm EXCEL II Kit non-cycle protocol with the protocol from a widely used sequencing kit, T7 Sequenase® (Amersham), with respect to these steps. Using the SequiTherm EXCEL II Kit,4 template denaturation is achieved by a 1-5 minute incubation at 95°C. The T7 Sequenase kit provides three different ways to denature dsDNA: alkaline denaturation followed by ethanol precipitation,5 alkaline denaturation without precipitation,6 and glycol plus heat.6 We have consistently generated the best T7 Sequenase data using the 60-minute alkaline denaturation/ethanol precipitation protocol (Figure 1). The simple heat denaturation step in the SequiTherm EXCEL II protocol is both faster and easier to perform than any of the T7 Sequenase methods. Similarly, the primer annealing and extension/termination steps are faster and easier to perform using the SequiTherm EXCEL II Kit. The total incubation time of these three steps using the SequiTherm EXCEL II Kit requires a maximum of 15 minutes, but can be completed in as little as 4 minutes