Sequencing at Fidelity Systems

     We have developed a robust procedure for high quality sequencing directly off BAC or PAC DNA templates. The sequencing reactions customized for BAC or PAC DNAs give long, high quality reads. The reaction is based on dye terminator sequencing chemistry and incorporates ThermoFidelase proteins and specially modified oligonucleotide ("Fimer") with greatly improved template-annealing characteristics.

     The technology is suitable both for closing gaps and low quality regions in working draft sequences obtained by shotgun sequencing of cloned human or animal DNAs, or for complete sequencing of unknown DNA cloned into BAC. The use of a single BAC preparation eliminates the necessity of storing and tracking thousands of plasmid clones and/or generating new libraries required by current finishing procedures. Finishing by direct sequencing results in a smaller number of reduced quality bases in final assemblies, as compared to a full shotgun, and solves many problems on difficult templates that are accumulated in shotgun projects with standard sequencing protocols.

     The procedures developed at Fidelity Systems are especially suitable to apply non-standard primers on BAC templates and attain very specific goals in high-quality sequencing. Extension of BAC sequencing with D-Strap(tm) technology allows for high-throughput sequencing of similar DNAs of related species or for precise description of pathological DNA modifications.

High Quality BAC End Sequencing 30 ng BAC with a T7 Fimer and TermoFidelase; 400 thermal cycles

 

Shotgun reads were downloaded from Trace archive (GenBank)   BAC containing mouse SEP15 gene was sequenced at FSI with Fimer and ThermoFidelase.

Direct sequencing of BAC templates with 'Alu' primers

Direct sequencing of BAC DNA with non-standard primers

 

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Covered by U.S. Patents 5,427,928, 5,656,463, 5,902,879, 6,548,251. Patents pending. Fidelase, ThermoFidelase, D-Strap and Fimer are trademarks of Fidelity Systems.