Generation of modified polymerases for improved accuracy in...

C - Chemistry – Metallurgy – 12 – N

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C12N 9/12 (2006.01) C12N 15/10 (2006.01) C12N 15/54 (2006.01)

Patent

CA 2720046

Provided are compositions comprising modified recombinant polymerases that exhibit branching fractions that are less than the branching fractions of the polymerases from which they were derived, or branching fractions that are less than about 25% for a phosphate-labeled nucleotide analog. Also provided are compositions comprising modified recombinant polymerases that exhibit closed polymerase/DNA complexes with increased stability relative to the parental polymerases. Also provided are compositions comprising modified recombinant polymerases that exhibit decreased rate constants relative to the parental polymerases. Provided are methods for generating polymerases with the aforementioned phenotypes. Provided are methods of using such polymerases to make a DNA or to sequence a DNA template.

L'invention porte sur des compositions comprenant des polymérases recombinantes modifiées qui présentent des fractions de ramification qui sont inférieures aux fractions de ramification des polymérases à partir desquelles elles sont issues, ou des fractions de ramification qui sont inférieures à environ 25 % pour un analogue nucléotidique marqué au phosphate. L'invention porte également sur des compositions comprenant des polymérases recombinantes modifiées qui présentent des complexes polymérase/ADN fermés avec une stabilité accrue par rapport aux polymérases parentales. L'invention porte également sur des compositions comprenant des polymérases recombinantes modifiées qui présentent des constantes de vitesse diminuées par rapport aux polymérases parentales. L'invention porte sur des procédés pour générer des polymérases avec les phénotypes mentionnés ci-dessus. L'invention porte sur des procédés d'utilisation de telles polymérases pour fabriquer un ADN ou pour séquencer un modèle d'ADN.

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