Sequencing Difficult DNA Templates

     Standard shotgun protocols often fail to produce satisfactory reads with so-called "difficult" templates, such as GC- and AT-rich samples, long simple mono- and di-nucleotide repeats, multiple inverted (hairpin) repeats, and regions with strong stop sites. Our sequencing technology solves many problems with difficult templates using modified protocols with ThermoFidelase and Fimers.

Examples of DNA Sequencing from Difficult Templates

 

Repeats

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     Sequencing through repeated and structured regions: simple nucleotide repeats, Pu and Py repeats.

 

Standard protocol     Modified reaction with ThermoFidelase

 

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Standard sequencing protocol     Modified protocol with ThermoFidelase

     Sequencing AT-rich templates: plasmids with cloned DNA of Plasmodium falciparum.

 

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Sequencing through long stable hairpins in a plasmid with simple nucleotide repeats using a modified protocol with ThermoFidelase.

 

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     Sequencing through structured regions: long GC rich hairpins (40 kb template)

 

Sequencing through structured regions: multiple hairpin repeats in pTAR25 (anti-HIV gene therapy construct).

 

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Sequencing through repeats in PAC's CCA-rich region.

 

Modified reaction with ThermoFidelase: closing of a long CT repeat in PAC RPCI-233D13.

 

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Selective sequencing of BAC repeats: getting single reads with multiple priming after treatment of the template DNA with a restrictase.

 

Stops

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Sequencing through strong STOPs in BAC templates

 

Standard sequencing protocol   Modified protocol with ThermoFidelase and a Fimer

     BAC with cloned DNA from Arabidopsis thaliana.

 

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Standard sequencing protocol   Modified protocol with ThermoFidelase and a Fimer

 

Standard sequencing protocol   Modified protocol with ThermoFidelase and a Fimer

     Stops in human DNA clones in BAC.

 

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Sequencing through strong STOPs in viral and bacterial DNAs.

Reading through stable hairpins in AAV-2 LTR DNA that stop standard sequencing of the template.             Standard sequencing protocol vs   Modified protocol with ThermoFidelase and a Fimer

 

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Standard sequencing protocol vs   Modified protocol with ThermoFidelase and a Fimer

     Sequencing through multiple strong stops (40 kb template DNA from Rhodobacter capsulatus).

 

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Standard sequencing protocol vs   Modified protocol with ThermoFidelase, a Fimer, and TopoTaq DNA polymerase

     Sequencing through strong stop (template DNA from Bifidobacterium cloned into pUC19).

 

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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.