High fidelity polymerases and uses thereof

C - Chemistry – Metallurgy – 12 – N

Patent

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Details

C12N 15/54 (2006.01) A61K 39/395 (2006.01) A61K 48/00 (2006.01) C07H 21/04 (2006.01) C07K 16/00 (2006.01) C12N 9/12 (2006.01) C12N 15/10 (2006.01) C12Q 1/68 (2006.01)

Patent

CA 2415767

The invention relates to a DNA and RNA polymerases which have increased fidelity (or reduced misincorporation rate). In particular, the invention relates to a method of making such polymerases by increasing or enhancing 3'- 5' exonuclease activity of a polymerase, for example, substituting the 3'-5' exonuclease domain of one polymerase with a 3'-5'exonuclease domain with the desired activity from another polymerase. The invention also relates to DNA molecules containing the genes encoding the polymerases of the invention, to host cells containing such DNA molecules and to methods to make the polymerases using such host cells. The polymerases of the invention are particularly suited for nucleic and synthesis sequencing, amplification and cDNA synthesis.

La présente invention concerne des ADN polymérases et des ARN polymérases présentant une fidélité accrue (ou à taux d'incorporation erroné réduit). Elle concerne, en particulier, un procédé de préparation de telles polymérases par augmentation ou activation de l'activité de 3'-5' exonucléase d'une polymérase, par exemple, en substituant le domaine 3'-5' exonucléase d'une polymérase par un domaine 3'-5'exonucléase caractérisé par l'activité souhaitée d'une autre polymérase. Par ailleurs, cette invention concerne des molécules d'ADN contenant des gènes codant les polymérases précitées, des cellules hôtes contenant de telles molécules d'ADN, ainsi que des méthodes d'incitation des polymérases à utiliser ces cellules hôtes. Les polymérases de l'invention sont particulièrement appropriées pour la synthèse d'acides nucléiques, le séquençage, l'amplification et la synthèse d'ADNc.

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