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United States Department of Agriculture

Agricultural Research Service

Title: Pcr-Based Reaction Setup for High-Throughput Cdna Library Sequencing on the Abi 3700 Automated DNA Sequencer

Authors
item Smith, Timothy
item Godtel, Renee
item Lee, Robert

Submitted to: Biotechniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 30, 2000
Publication Date: N/A

Interpretive Summary: We describe a method that we developed for preparation of template and sequencing of genes using the new automated sequencer called the ABI 3700. These machines have rapidly become the dominant technology for sequencing of human and other genes and genomes, and new and cheaper approaches to sequence set-up are needed to make them economical and efficient.

Technical Abstract: Numerous large-scale sequencing projects are now being conducted or considered that rely on the latest capillary electrophoresis-based automated sequencing system from Applied Biosystems, the ABI 3700. Due to the electrokinetic injection protocol and the physical properties of the capillaries and polymer employed, reaction setups that have been optimized for the gel-based ABI 377 systems do not necessarily perform to expectations in the capillary system. We have found this is especially the case when sequencing PCR products. We describe a rapid, inexpensive, and reliable method for preparing template and sequencing reactions for application to high-throughput sequencing of the coding strand of cDNA libraries on the ABI 3700 instrument. The basic setup has been very successful in generating expressed sequence tag (EST) data on the coding strand. We have produced approximately 50,000 sequences using this method, with an overall success rate of 75-80%. This approach does not work for sequencing the noncoding strand using vector primers, presumably due to slippage of the polymerase during amplification of the clones leading to variation in the length of the poly T region at the end of the clone. However, this limitation can be circumvented using anchored poly T primers to generate 3 prime end sequence. The total cost of all materials and reagents, on a per sequence basis, is approximately equal to the per clone cost of commercially available automated miniprep kits.

Last Modified: 11/28/2014
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