Pfu replication accessory factors and methods of use

Abstract

This invention provides isolated polynucleotides that encode replication accessory factors. The invention also provides novel DNA replication accessory factors, which have been isolated and purified from the hyperthermophilic archaea Pyrococcus furiosus. The invention also provides various methods of enhancing a nucleic acid polymerase reaction comprising the addition of the replication accessory factors to the reaction. This invention further provides methods of synthesizing, amplifying, and mutagenizing nucleic acids of interest employing the replication accessory factors. This invention also provides kits comprising, at least one of the replication accessory factors. This invention also provides kits useful for various methods that comprise at least one replication accessory factor.

Description

RELATED APPLICATION INFORMATION[0001]This application is a divisional application of U.S. application Ser. No. 10 / 828,924, filed Apr. 20, 2004 (now U.S. Pat. No. 7,442,766), which is a continuation of U.S. application Ser. No. 09 / 626,813, filed Jul. 27, 2000 (abandoned), which claims the benefit of U.S. Provisional Application Ser. No. 60 / 146,580, filed Jul. 30, 1999. Each of these prior application is herein incorporated by reference in its entirety.BACKGROUND AND SUMMARY OF THE INVENTION[0002]The invention relates to the field of replicating, amplifying, and sequencing nucleic acids. Further, this invention relates to novel proteins that enhance the activity of the polymerases.[0003]In vitro polymerization techniques have enormously benefited the fields of biotechnology and medicine. The ability to manipulate nucleic acids with polymerization reactions greatly facilitates techniques ranging from gene characterization and molecular cloning (including, but not limited to sequencing,...

AI Technical Summary

Benefits of technology

[0002]The invention relates to the field of replicating, amplifying, and sequencing nucleic acids. Further, this invention relates to novel proteins that enhance the activity of the polymerases.

[0003]In vitro polymerization techniques have enormously benefited the fields of biotechnology and medicine. The ability to manipulate nucleic acids with polymerization reactions greatly facilitates techniques ranging from gene characterization and molecular cloning (including, but not limited to sequencing, mutagenesis, synthesis and amplification of DNA), determining allelic variations, and detecting and screening of various diseases and conditions (e.g., hepatitis B).

Problems solved by technology

Multiple cycles of PCR result in an exponential amplification of the DNA template.

Unfortunately, PCR has limitations.

But, Taq polymerase has a fundamental drawback because it does not have 3′ to 5′ exonuclease activity and, therefore, cannot excise incorrect nucleotides added to the ends of the amplified products.

Due to this limitation, the fidelity of Taq-PCR reactions typically have suffered.

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