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US 6,783,941 B2Granted
Method for producing a polynucleotide library in vitro by mismatch repair of heteroduplexes
AssigneeNovozymes A/S
InventorJesper Vind
Life of this patent
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Abstract
A method for making polynucleotide libraries that comprise new recombined polynucleotides by using mismatch repair enzymes or proteins to shuffle at least two variants of the same polynucleotide or at least two homologous polynucleotides through a Recombinatorial Chain Reaction, RCR.
Claims17
A method for forming a plurality of recombined homologous double-stranded polynucleotides from at least two homologous double-stranded template polynucleotides, said method comprising the steps of:
- a) providing a solution comprising at least two non-methylated homologous double-stranded template polynucleotides and one or more thermostable mismatch repair protein(s);
- b) denaturing the template polynucleotides into single-stranded polynucleoides by increasing the temperature of the solution;
- c) annealing the different single-stranded polynucleotides by lowering the temperature of the solution, wherein heteroduplexes are formed;
- d) allowing the thermostable mismatch repair protein(s) to repair nucleotide mismatches in the heteroduplexes, wherein recombined new duplexes are formed;
- and
- e) repeating steps b) through d) at least once;
- wherein the new duplexes of step d) serve as new template polynucleotides in step b) in each repetition.
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Dependencies are inferred from the claim text.