ThermoFidelase I for DNA sequencing

ORDERING INFO Cat. No Size, ul A016 100 A056 500 A106 1000 A156 5x1000 CONTACT INFO 301-527-0804 (tel) 301-527-8250 (fax) fsi1@fidelitysystems.com LINKS Genomic DNA U Oklahoma-ACGT UAB & PE-ABI-ACGT Capillary Sequencer ABI User's Manual-p.2-27 BAC, PAC, P1, Cosmids DAF-Johns Hopkins U Intron/Exon Structure Iowa U Human-Mouse Comparison Johns Hopkins U Cereal, Maize BACs Purdue U U of Arizona GC-rich Samples ABC-Imperial C@Charing Cross ABI-Sequencing Troubleshooting Stop Regions Washington U St. Louis-GSC Multiple STOPs

PRODUCT DESCRIPTION ThermoFidelase I is a HyperStable protein that significantly enhances the success rate of DNA sequencing reactions. It enzymatically unlinks and denatures plasmid DNA, helps DNA polymerase to pass through strong secondary structures and protects DNA from thermal decomposition. ThermoFidelase I has been successfully used to sequence many 'difficult' DNA samples that for different reasons could not been sequenced previously. The addition of ThermoFidelase to regular sequencing reactions results in a lower rate of errors and increases the accuracy of base calling. Use of Thermofidelase I results in an increase in the correct and unambiguous read length, improved peak resolution on traces, and stronger signal intensity on gel images and autoradiograms. ThermoFidelase is compatible with isothermal and cycle sequencing using fluorescent or radioactive labels and automated or manual procedures. CYCLE SEQUENCING For one 20 ul reaction, mix 1 ul of ThermoFidelase I with the template DNA and follow the procedure recommended for your cycle sequencing kit. DIRECT AUTOMATED SEQUENCING OF BACTERIAL GENOMIC DNA Major breakthrough in DNA sequencing technology From oral and poster presentations of PE Applied Biosystems (developed and used by Cheryl Heiner and Kathryn Hunkapiller) and U Oklahoma (Drs. Bruce Roe and Sandra Clifton group) at the Genome Sequencing and Analysis Conference (Hilton Head, Sept 1997). 16 ul BigDye Terminator Mix 1 ul ThermoFidelase I 15-30 pmol Primer (18-24 mer) 3-6 ug Genomic DNA 40 ul Cycle: 95 C - 5 min (95 C - 30 sec) 45 x (55 C - 20 sec) (60 C - 4 min) 4 C - hold Recent experiments with Genomic DNA Templates, Roe-Lab 1x reaction, Modified Bacterial Thermo-Cycle Conditions and comprehensive discussion is now available from UO-ACGT. The high-sensitivity procedure for cycle sequencing of Genomic DNA using Capillary Sequencer is in ABI User's Manual on page 2-27 (P/N 903565 rev B). WASHINGTON UNIVERSITY PROTOCOL This approach has proven successful in sequencing through stop regions of M13 or pUC subclones, and PCR products It was presented by Dr. Elaine Mardis at the CSH Meeting 'Genome Mapping and Sequencing' (p.140) and is available from WUSTL-GSC. SEQUENCING WITH NON-THERMOSTABLE DNA POLYMERASE For one reaction, mix 1 ul of ThermoFidelase I with template DNA and add other reagents of sequencing reaction (buffer, primer) but do not add DNA polymerase. Incubate at 95 C for 5 minutes and cool to room temperature. Add DNA polymerase and follow the procedure recommended for your sequencing kit. FOR RESEARCH USE ONLY Covered by U.S. Patents 5,427,928, 5,656,463. Patents pending. Fidelase, ThermoFidelase and HyperStable are trademarks of Fidelity Systems. ThermoFidelase development is supported in part by the National Institutes of Health (NIGMS)

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