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.
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
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
stop regions of M13 or pUC subclones, and PCR products
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.