ThermoFidelase 2 Kit with Fimers, No Errors BAC sequencing, Whole Genome Direct Sequencing of the Hyperthermophile Methanopyrus kandleri with D-Strap Fimers

ThermoFidelase 2 Kit with Fimers

Recent story in The New York Times describes a critical role of the ASPM gene in brain development. A team of scientists discovered an accelerated evolution of the ASPM gene controlling brain size in primate lineage (see publ.). A technology behind the discovery is the TAR-BAC capture of any gene from human and other genomes developed by Larionov's group. All captured 65-kb genes were sequenced with a common set of 384 D-Strap Fimers according to ThermoFidelase sequencing specifications. The complete ASPM genes from chimp, gorilla, orangutan and macaque do not contain sequencing errors, cloning or PCR artifacts. ThermoFidelase sequencing specifcations were first applied for a gene from cancer patient.

ORDERING INFO
Standard Fimer

Cat. No Size, seq.rxns

B016 100

B106 1000

B156 5000

Custom Fimer

Cat. No Size, seq.rxns

B016c 100

B106c 1000

Gene Knock Out ID
Standard Fimer

Cat. No Size, seq.rxns

C016 100

C106 1000

C156 5000

Custom Fimer

Cat. No Size, seq.rxns

C016c 100

Cat. No Size, seq.rxns

ThermoFidelase 2 is a thermostable DNA unlinking enzyme (type IB DNA topoisomerase). During initial heating of the reactions it removes topological linkage between two strands of plasmid DNA. Because of the topological constraint in the DNA produced by unlinking, the unlinked plasmid strands cannot reform a normal double helix at the lower temperature used for primer annealing and extension. This results in an increased availability of targets for primer annealing and consequently a higher yield of cycle sequencing reaction products.

Fimers are primers with proprietary chemical modifications that inhibit unwanted reactions during cycle sequencing (e.g., primer-dimer artifacts or non-specific PCR). Specially designed Fimers have been successfully used for high throughput sequencing of difficult templates and highly sensitive reactions from minimal amount of templates (1 ng plasmid, 10 ng BAC, 100 ng microbial genomic DNA). Fimers can be designed for any sequencing applications. Kit B106 contains Forward and Reverse Fimers optimized for plasmids containing inserts of AT-rich Plasmodium falciparum DNA.

Reagent Quantity per reaction
Plasmid DNA 50 - 200 ng
ThermoFidelase 2 0.1 uL
Fimer 1 uL
Dye Terminator Mix 2 uL
dH₂O q.s.
Volume 5 uL


  
For 96 well plate, mix the following
 ThermoFidelase 2 10.6 uL
Fimer 106 uL
Dye Terminator Mix 212 uL
 
Mix well by pipetting.
Dispense 3.1 uL per well.
Add 2 uL of corresponding template in each well. Seal the plate.
Spin the plate briefly before placing in the thermocycler.
   Use the following cycling conditions:
 Heat the plate at 95ーC for 2 minutes
 Repeat the following for 30 cycles*:
95ーC for 5 seconds
50ーC for 30 seconds
60ーC for 4 minutes
 Rapid thermal ramp to 4ーC and hold until ready to purify


* Up to 100 cycles is recommended when sequencing from less than 50 ng of plasmid DNA

FOR RESEARCH USE ONLY 
 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. ThermoFidelase, D-Strap and Fimer development is supported in part by the National Institutes of Health (NIGMS) and US Department of Energy