Clontech Laboratories, Inc.

Helmut Wurst

Gregory D Davis

Ningyue Zhao

Zhi-Hao Qiu

Methods are provided for evaluating the fidelity of a given set of polymerase chain reaction conditions. In the subject methods, a template polydeoxyribonucleotide is amplified under the to be evaluated polymerase chain reaction conditions, where the template polydeoxyribonucleotide includes a pseudo restriction endonuclease restriction site. The resultant amplified product population is then contacted with the corresponding restriction endonuclease and resultant cleavage products, if any, are detected. The fidelity of the polymerase chain reaction conditions is then derived from the detected cleavage products (or absence thereof). Also provided are kits for use in practicing the subject methods. The subject methods are suited for determining the fidelity of a given polymerase under PCR conditions, and are particularly suited for determining the fidelity of a thermostable polymerase under PCR conditions.

Methods and kits for determining the fidelity of polymerase chain reaction conditions

Enzyme compositions, kits comprising the same and methods for their use in one-step RT-PCR are provided. The subject enzyme compositions at least include a mutant thermostable DNA polymerase and a mutant reverse transcriptase. In preferred embodiments, the mutant thermostable DNA polymerase is an N-terminal deletion mutant of Taq polymerase and the mutant reverse transcriptase is a point mutation mutant of MMLV-RT. The subject kits, in addition to the above described mutant thermostable DNA polymerase and mutant reverse transcriptase, at least include one of, and usually both of, dNTPs and a buffer composition, where the subject kits may further include additional reagents, including nucleic acids, a thermostabilizing agent, a glycine based osmolyte and the like. In practicing the subject methods, a reaction mix that at least includes template RNA, the above described mutant polymerase and reverse transcriptase, dNTPs, buffer, and nucleic acid primers is prepared. The resultant reaction mixture is maintained at a first set of reverse transcription conditions and then a second set of PCR conditions, whereby amplified amounts of DNA from a template RNA(s) are produced.

One step RT-PCR methods, enzyme mixes and kits for use in practicing the same

A thermostable enzyme having polymerase activity and substantially no nuclease activity is provided. The thermostable enzyme is characterized by comprising a sequence of nine amino acid residues at least proximal to the N-terminus that has less than 50% but greater than 40% amino acid sequence identity with residues 280 to 288 of the naturally occurring Thermus aquaticus polymerase. The subject enzyme finds use in a variety of different application in which polynucleotides are enzymatically produced, particularly in PCR based applications.

Thermostable polymerase

Methods and compositions are provided for performing high fidelity polymerase chain reactions. In the subject methods, primer extension products are produced with a low error frequency rate through the use of at least one of unequal concentrations of dNTPs or a melting point reducing agent. Also provided are kits and reagent mixtures for use in the subject methods. The subject invention finds use in a variety of applications, particularly in applications where high fidelity PCR is desired.

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Cupertino, CA, United States of America

Helmut Wurst