Sequencing at Fidelity Systems

Crude, Uncharacterized, Mixed DNA...

     Fidelity Systems offers sequencing off crude, uncharacterized, or mixed DNA samples, or samples with a small amount of template. We can perform, if possible, direct sequencing off template DNA without purification or use amplification of the templates with Phi29 DNA polymerase and Phi29 Random Hexamer Primers.

Direct Sequencing Plasmids in Cell Cultures (E. coli; 1 l overnight culture; Fimer; ThermoFidelase; 400 cycles)


pUC

 

pGEM-3Zf(+)

 

M. Kandleri
AV19 DNA
cloned into pUC19
library

 

Plasmodium falciparum
DNA cloned
into pFKBP50


Standard sequencing protocol

 

Modified protocol
with ThermoFidelase,
a Fimer, and
TopoTaq DNA polymerase

 

Modified protocol;
E.coli cells; TopoTaq
DNA polymerase mix

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


 

     Standard high-quality read off plasmid template DNA amplified directly from E.coli cells with Phi29 DNA polymerase and Phi29 Random Hexamer Primers.



Applications of Direct Sequencing off Genomic DNA

     Sequencing uncharacterized genomic samples uses D-Strap strategy that includes design degenerate primers targeting evolutionary conserved regions in genomic DNA. FSI offers sequencing of the following types of targets:
     - rRNA genes (universal)
     - protein genes (variable)
     - tRNA and hot spots of genome plasticity

     We also offer sequencing of flanking regions to the known sequences off genomic DNA.



plasmid:           gtcgagatgcctgacccggaaccgtgatcgccgatcacAGCTTGCGGCCGCAC
         	          |||||||||||||||||||||||||||||||||||||| 
M.kandleri:	 gtcgagatgcctgacccggaaccgtgatcgccgatcacGTGCGGAGGTTAGCC 

Sequencing Beyond
Cloned Genes:


Plasmid vs. Genomic
DNA Templates


Universal Fimer
Selective Fimer A

 

Selective Fimer B

     Deconvolution of gene homologs: universal vs. selective Fimers for each gene copy tufA / tufB E.coli B.


E.coli control

 


Sample AL-19 from Kenya

 

Chain A, Elongation Factor Complex Ef-TuEF-Ts

 

translation elongation factor Tu.A

     Discovery and direct sequencing of new protein genes with Fimers for evolutionary conserved proteins.

 


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.
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