Fast Plasmid DNA Sequencing Using a Thermal Cycler and High Temperature Alkali Denaturation

This report describes a modification of the Chen and Seeburg plasmid sequencing procedure (2). Chen and Seeburg demonstrated that the collapse of closed covalent circular plasmid DNA into networked DNA favored doublestranded sequencing; however, the method was sensitive to genomic DNA and RNA contamination. Complete alkaline denaturation (not thermal) and RNA degradation were essential for high resolution sequencing and achieving a low background. Current modifications of this protocol work well only when high-quality double-stranded plasmid is obtained as a template (1). This involves multiple steps to remove genomic DNA and RNA contamination by either LiCl precipitation, RNase digestion, CsCl ultracentrifugation, specialized resins or polyethylene glycol purification (3,4). These are time-consuming, or they necessitate the use of expensive kits for plasmid preparation if quite a few plasmids are to be sequenced. Our results show that a 10min plasmid preparation step when combined with a high temperature alkali denaturation step similar to that described by Musich and Chu (3) produces high resolution sequencing results. Incubation of reaction components in a thermal cycler and utilization of tube strips permit rapid throughput under identical reaction conditions and greatly enhances reproducibility. Plasmids in XL2-Blue (Stratagene, La Jolla, CA, USA) are grown in LB medium with appropriate antibiotics overnight at 37°C and are processed by the 10-min method of Zhou et al. (5). Briefly, 1.5 mL of an overnight culture are collected at 15 000× g for 20 s, 50–100 μL of supernatant should be allowed to remain for resuspending the bacterial pellet by vortex mixing and 300 μL of lysis buffer (0.1 N NaOH, 0.5% sodium dodecyl sulfate in 1× TE buffer [10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0]) are added to the suspension. After 5 s of vortex mixing, 150 μL of 3 M Na acetate (pH 5.2) are added and vortex mixed for 5 s. It is important to denature and neutralize the reaction without delay by vortex mixing to avoid the genomic DNA contamination. After centrifugation at 15 000× g for 2–3 min, the supernatant is transferred to 900 μL of ethanol and mixed well, and the plasmid is recovered at 15 000× g for 2–3 min and rinsed twice in 70% ethanol. The residual ethanol is removed by pipetting and air-drying. Then the steps in Table 1 are followed. We found there is no difference in the sequencing result from RNase Adigested, phenol-purified plasmids and CsCl-purified plasmids (data not shown) if the high temperature alkali denaturation step is used instead of the room temperature alkali denaturation originally recommended. Results are more reproducible if a thermal cycler and PCR tube strips are used for maintaining precise temperature control and for making the various reactions easier to manipulate during denaturation, chain elongation/termination and loading, thus saving time and the possibility of handling errors. Double-stranded plasmid templates can be prepared and sequenced by this method through the gel electrophoresis step in a single working day, and the autoradiographic