Method for in vitro molecular evolution of protein function
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Abstract
The invention provides a method for generating a polynucleotide sequence or population of sequences from parent single stranded polynucleotide sequences encoding one or more protein motifs, comprising the steps of a) providing single stranded DNA constituting plus and minus strands of parent polynucleotide sequences; b) digesting the single stranded polynucleotide sequences with a nuclease other than DNase I to generate populations of single stranded fragments; c) contacting said fragments generated from the plus strands with fragments generated from the minus strands and optionally, adding primer sequences that anneal to the 3′ and 5′ ends of at least one of the parent polynucleotides under annealing conditions; d) amplifying the fragments that anneal to each other to generate at least one polynucleotide sequence encoding one or more protein motifs having altered characteristics as compared to the one or more protein motifs encoded by said parent polynucleotides.
17 Citations
14 Claims
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1-7. -7. (canceled)
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8. A method for generating a polynucleotide sequence or population of sequences from parent single stranded polynucleotide sequences encoding one or more protein motifs, comprising the steps of:
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a) providing single stranded polynucleotide sequences constituting plus and minus strands of parent polynucleotide sequences;
b) digesting the single stranded polynucleotide sequences with an exonuclease to generate a first population of single stranded fragments comprising fragments generated from the plus strands and a second population of single stranded fragments comprising fragments generated from the minus strands, said second population being separate from said first population;
c) contacting said first population of single stranded fragments with said second population of single stranded fragments, whereby annealing occurs between said first population of single stranded fragments and said second population of single stranded fragments;
d) amplifying the fragments that anneal to each other to generate at least one polynucleotide sequence encoding one or more protein motifs having altered characteristics as compared to the one or more protein motifs encoded by said parent polynucleotides. - View Dependent Claims (9, 10, 11, 12, 13, 14)
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Specification