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Automated Fluorescent-Tag Cycle Sequencing

Application of the RoboCycler Gradient 96 temperature cycler

Douglas Drake
Stratagene Cloning Systems, Inc.

Stratagenes RoboCycler Gradient 96 temperature cycler*,# with Hot Top Assembly can be used to perform fluorescent-tag cycle sequencing using the ABI PRISM dye terminator cycle sequencing ready reaction kit. The new Hot Top Assembly eliminates the need for oil or wax sample overlays that hinder the loading of cycle sequencing reactions onto polyacrylamide gels. With unmatched well-to-well temperature accuracy, the RoboCycler Gradient 96 temperature cycler ensures the even and reproducible reaction conditions that are essential for achieving uniform fluorescent-tag signals and better read accuracy.

Cycle sequencing, using fluorescent dye-tagged terminators or primers, has become the standard protocol for use with automated sequencing devices.1 The fluorescent dye-labeled cycle sequencing products are loaded onto a vertical denaturing polyacrylamide gel and separated by gel electrophoresis. Automated gel reading systems contain one or more lasers that excite the fluorescent tag and sensors that detect fluorescent emissions within each lane. These systems can typically detect and distinguish different fluorescent tags on the basis of emission wavelength and quantitate the strength of fluorescent signals at each reaction termination point. By detecting the fluorescent-dye tags along the electrophoretic axis of the gel, the instrument can automatically determine the correct sequence of the reaction template with greater than 98% accuracy.2

PE Applied Biosystems have found that, for their ABI PRISM 377 and 373 DNA sequencers, poorly calibrated temperature cyclers can have a dramatic effect on the sensitivity, specificity and reproducibility of cycle sequencing reactions.3 When multiple fluorescent tags are used for cycle sequencing, uniform signal strength among the different tags is especially important for scan and base-calling accuracy. A temperature cycler block that does not produce equal cycling conditions in each well may not incorporate the dideoxy nucleotide reaction terminators at a uniform rate between sample wells. Incorporation of fluorescent tags that is not uniform results in uneven band intensities, which are difficult to scan. Thus, the sequence of the resulting cycle sequencing product cannot be determined accurately.

figure 1

Stratagenes line of RoboCycler temperature cyclers have unmatched well-to-well temperature uniformity during PCR amplifications (figure 1). The addition of the new Hot Top Assembly, which is available for both gradient and nongradient RoboCycler 40 and 96 models, eliminates the need for an oil or wax overlay on reactions. The Hot Top Assembly prevents sample evaporation even for the small reaction volumes that are typically used in cycle sequencing reactions. We determined the accuracy with which the RoboCycler Gradient 96 temperature cycler with a Hot Top Assembly can perform fluorescent-dye cycle sequencing reactions.

Comparison of Temperature Cyclers

Cycle sequencing experiments were performed using both Stratagenes RoboCycler Gradient 96 temperature cycler with Hot Top Assembly and the GeneAmp PCR System 9600 cycler. The ABI PRISM dye terminator cycle sequencing ready reaction kit was used to sequence the kits control plasmid, the pGEM -3Zf(+) vector, using the (-21) M13 forward primer. Both experiments were performed according to the manual supplied with the ABI PRISM sequencing kit. Following the completion of cycle sequencing, the 20-l reaction volumes were ethanol precipitated, dried and resuspended in 6 l of gel loading buffer. Two-microliter aliquots of both sets of reactions were loaded onto a 4.8% Gene Page Plus urea polyacrylamide gel and run on an ABI PRISM 377 DNA sequencer.

Results

figure 2, panel A

figure 2, panel B

The ABI PRISM 377 sequencer automatically scans gels to identify lanes and detect the fluorescent-dye terminators within each lane. The lane tracking was corrected to pass through the center of each lane, where the band fluorescence is maximal. Electropherograms (figure 2, panel A, panel B) were generated for both lanes showing the relative fluorescent intensities of each of the four dideoxy nucleotide reaction terminators (A-Dye Terminator, C-Dye Terminator, G-Dye Terminator and T-Dye Terminator) labeled with a different fluorescent tag. As the excitation laser scans along the electrophoretic axis of the gel, it excites and detects the emission from each tag at each cycle sequencing termination point within the gel. Each of the four fluorescent tags are represented within the electropherogram as different emission line plots, green for A-Dye Terminator, red for T-Dye Terminator, black for G-Dye Terminator and blue for C-Dye Terminator. The four emmision line plots are superimposed to show their spacial relationship within the gel along the electrophoretic axis. The base sequence of the template, as determined by ABI PRISM software, is written above the emission plot lines. The relative number of the nucleotide from the primer is indicated. figure 2 panel A shows the sequencing histogram generated using the RoboCycler Gradient 96 temperature cycler with Hot Top Assembly, showing read length to 745 bases. figure 2 panel B shows the results generated from the cycle sequencing reactions run on the GeneAmp PCR System 9600 cycler, showing read length to 720 bases. The sequence read from reactions cycled on the RoboCycler Gradient 96 temperature cycler required 2 hours and was 1% inaccurate or unassignable. The sequence read from reactions cycled on the GeneAmp PCR System 9600 cycler (using the manufacturers protocol) took over 20 minutes longer than the reactions cycled with the RoboCycler unit. In addition, the sequence generated from reactions cycled on the GeneAmp system was 2% inaccurate or unassignable. The intensities of the fluorescent-tag signals were more uniform for the reactions performed on the RoboCycler Gradient 96 temperature cycler with Hot Top Assembly than for the reactions performed on the GeneAmp PCR System 9600 cycler. This difference in uniformity may have caused the difference in accuracy of base recognition between reactions performed on the two instruments.

Conclusions

With its unmatched well-to-well temperature accuracy, the RoboCycler Gradient 96 temperature cycler with Hot Top Assembly performs fluorescent-dye cycle sequencing reactions accurately and reproducibly. The new Hot Top Assembly for both gradient and nongradient RoboCycler 96 temperature cyclers eliminates the need for oil or wax overlays on reactions during PCR cycle sequencing, which makes loading samples onto automated DNA sequencing instruments quick and easy.

REFERENCES

  1. Adams, M.D., Fields, C., and Venter, J.C., eds. (1994) In Automated DNA Sequencing and Analysis. Academic Press, San Diego, California.

  2. Applied Biosystems. (1993/1994) In ABI Product Catalog, p.144. Foster City, California.

  3. Applied Biosystems. (1995) In ABI PRISM, Comparative PCR Sequencing, p. 18. Foster City, California.


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