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Serial Analysis of Gene Expression Using ABI PRISM DNA Sequencers and BigDye Terminators

h degree of accuracy that transcript analysis of short 9- or 10-bp sequences demands. This accuracy, coupled with the sensitive detection capacity of the ABI PRISM 3100 or 3700 DNA sequencers, ensures a fast and accurate read. BigDye Terminator v 3.0 enables even greater signal uniformity for samples that show a tendency toward signal loss in longer fragments.

Complete Automation

Both the 3100 and 3700 DNA sequencers are designed for more than 24 hours of unattended operation. Human intervention is required only to place the 96- or 384-well sample trays on the instrument, and to replenish the polymer supply. All other steps, including capillary filling, sample loading, capillary rinsing, data extraction, and analysis are completely automated.

SAGE Sequence Protocol

In experiments using typical sequencing methods, along with the ABI PRISM 3100 Genetic Analyzer and 3700 DNA Analyzer for sequencing, Applied Biosystems scientists produced reproducible and accurate ditag sequences.

Samples were prepared from mRNA derived from a cell line, Mcf-7 (human breast adenocarcinoma). A sequence content of approximately 26 ditags was obtained using both the standard protocol with POP-5 polymer on the 3700 DNA Analyzer, and the Rapid Read protocol on the 3100 Genetic Analyzer. For optimal sequence efficiency, the insert length should be sufficient to fully utilize the length-of-read for the instrument platform and BigDye chemistry.

Throughput

Throughput is an important consideration when choosing a platform for sequence analysis. The elimination of gel tracking and other problems that can arise from slab-gel sequencing greatly improves the sequencing capacity of the average research laboratory. Throughput requirements depend on the quantity of tags to be analyzed. ABI PRISM DNA sequencers provide this flexibil
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