Process for preparing variant polynucleotides
AssigneeDsm IP Assets B.V.
InventorsRoelof Ary Lans Bovenberg, Richard Kerkman
Life of this patent
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Abstract
The present invention discloses a process for the preparation of variant polynucleotides using a combination of mutagenesis of a starting population of polynucleotides and recombination of the mutated polynucleotides. The process comprises the steps of subjecting a population of polynucleotides to (a series of) two (or more) separate PCR's comprising a first PCR with a forward mutation-specific primer for a position to be mutated and a reverse universal primer and a second PCR with a forward universal primer and a reverse mutation-specific primer for a position to be mutated. The products of the (series of) two (or more) PCR's are assembled by a polymerase, preferably in one tube.
Claims13
A process for preparing a population of variant polynucleotides comprising at least one polynucleotide having a desired property, comprising:
- (a) subjecting a starting population of polynucleotides comprising at least two different mutants of a parental polynucleotide to separate PCR's to result in products of the PCR's, wherein the at least two different mutants were selected for the desired property:
- (i) a first set of at least two PCR's, which are performed separately from each other, with each PCR using a forward mutation-specific primer which hybridizes to a mutated position in a mutant of the starting population and a reverse universal primer to generate a first common end for the first set of PCR's,
- (ii) a second set of at least two PCR's, which are performed separately from each other, with each PCR using a forward universal primer to generate a second common end for the second set of PCR's and a reverse mutation-specific primer which hybridizes to a mutated position in a mutant of the starting population, and
- (iii) optionally a third or more PCR with suitable forward and reverse primers;
- (b) assembling the resulting products of the PCR's using a polymerase to obtain said population of variant polynucleotides, wherein the variant polynucleotides differ in at least one position from the parental polynucleotide;
- and
- (c) optionally amplifying the population of variant polynucleotides.
A process for preparing a population of variant polynucleotides comprising at least one polynucleotide having a desired property, comprising:
- (a) subjecting a parental polynucleotide to mutagenesis to provide mutants;
- (b) screening the mutants to provide at least two different mutants selected for the desired property as a starting population of polynucleotides;
- (c) subjecting the starting population of polynucleotides comprising the at least two mutants to separate PCR's to result in products of the PCR's:
- (i) a first set of at least two PCR's, which are performed separately from each other, with each PCR using a forward mutation-specific primer which hybridizes to a mutated position in a mutant of the starting population and a reverse universal primer to generates a first common end for the second set of PCR's,
- (ii) a second set of at least two PCR's, which are performed separately from each other, with each PCR using a forward universal primer to generate a second common end for the second set PCR's and a reverse mutation-specific primer which hybridizes to a mutated position in a mutant of the starting population, and
- (iii) optionally a third or more PCR with suitable forward and reverse primers;
- (d) assembling products of the PCR's using a polymerase to obtain said population of variant polynucleotides, wherein the variant polynucleotides differ in at least one position from the parental polynucleotide;
- and
- (e) optionally amplifying the population of variant polynucleotides.
Dependencies are inferred from the claim text.