C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
C12N 15/54 (2006.01) C12N 9/12 (2006.01) C12N 15/70 (2006.01) C12P 19/34 (2006.01) C12Q 1/68 (2006.01)
Patent
CA 2174944
The invention relates to a substantially pure thermostable DNA polymerase from Thermotoga neapolitana (Tne) and mutants thereof. The Tne DNA polymerase has a molecular weight of about 100 kilodaltons and is more thermostable than Taq DNA polymerase. The mutant Tne DNA polymerase has at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3' 5' exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5' 3' exonuclease activity of said DNA polymerase; (3) a third mutation in the O helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides. The present invention also relates to the cloning and expression of the wild type or mutant Tne DNA polymerase in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes. The Tne DNA polymerase of the invention may be used in well-known DNA sequencing and amplification reactions.
La présente invention concerne une ADN-polymérase thermostable sensiblement pure provenant de Thermotoga neapolitana (Tne) et leurs mutants. L'ADN-polymérase Tne présente un poids moléculaire d'environ 100 kilodaltons, mais est plus thermostable que l'ADN-polymérase Taq. L'ADN-polymérase mutante Tne présente au moins une mutation appartenant au groupe constitué de: (1) une première mutation qui réduit sensiblement ou élimine l'activité exonucléase 3' ? 5' de ladite ADN-polymérase; (2) une seconde mutation qui réduit sensiblement ou élimine l'activité exonucléase 5' ? 3' de ladite ADN-polymérase; (3) une troisième mutation au niveau de l'hélice O de ladite ADN-polymérase rendant ladite ADN-polymérase non discriminante par rapport aux bidésoxynucléotides. L'invention concerne également le clonage et l'expression de l'ADN-polymérase Tne de type sauvage ou mutant chez E. coli, des molécules d'ADN contenant le gène cloné, et des cellules hôtes exprimant lesdits gènes. L'ADN-polymérase Tne de la présente invention convient avantageusement aux réactions bien connues de séquençage et d'amplification de l'ADN.
Chatterjee Deb K.
Hughes A. John Jr.
Invitrogen Corporation
Mbm & Co.
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