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
C106c	1000

Plates with Custom Fimers

Cat. No	Size, seq.rxns
D016	96
D056	5*96

Plasmodium falciparum
TIGR

Methanopyrus kandleri
(2002) PNAS

No Errors BAC sequencing with D-Strap Fimers NCI, NIH
(2004) PNAS coming soon

Closing the Gaps on Human Chromosomes (2004) Genome Research 14, 239-246

Direct BAC Sequencing with ThermoFidelase and Fimers
(2004) BACs 1, 295-308
(2004) BACs 1, 221-230

Production of Fimers for Whole Genome Direct Sequencing
(2004) Oligonucleotide Synthesis. Methods and Applications. 291-304

CONTACT INFO
301-527-0804 (tel)
301-527-8250 (fax)
fsi1@fidelitysystems.com

PRODUCT DESCRIPTION
The B016 kit contains ThermoFidelase 2 and two Fimers for end sequencing from plasmid templates. It is designed for high throughput sequencing in 5 uL volume reactions with reduced consumption of dye terminator premix. Other versions of ThermoFidelase 2 kits with Fimers are designed for BAC, phage or genomic DNA sequencing.

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.

KIT CONTENT

110 uL ThermoFidelase 2

550 uL Forward Fimer
5'-GGGTTTTCCCAGTCACGACGTTGTA-3'

550 uL Reverse Fimer
5'-GGAAACAGCTATGACCATGATT-3': 5'-GGGTTTTCCCAGTCACGACGTTGTA-3'; 5'-GGAAACAGCTATGACCATGATT-3'

MATERIALS SUPPLIED BY THE USER
Template DNA, Dye Terminator Ready Reaction Mix, Deionized H₂O

CYCLE SEQUENCING
For sequencing of less than 96 samples, use the following table for calculation of the amount of each component

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°C for 2 minutes

Repeat the following for 30 cycles*:
95°C for 5 seconds
50°C for 30 seconds
60°C for 4 minutes

Rapid thermal ramp to 4°C and hold until ready to purify

* Up to 100 cycles is recommended when sequencing from less than 50 ng of plasmid DNA: 95°C for 5 seconds; 50°C for 30 seconds; 60°C for 4 minutes

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