Process for obtaining recombined nucleotide sequences in vitro, libraries of sequences and sequences thus obtained
First Claim
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1. A ligase-mediated method of in vitro recombination, comprising:
- providing oligonucleotide cleavage fragments derived from each of at least two different polynucleotides of a polynucleotide library;
hybridizing the cleavage fragments to an assembly template;
ligating together the hybridized fragments that have adjacent ends with a ligase;
denaturing the ligated fragments from the assembly template; and
repeating at least the hybridizing, ligating and denaturing steps multiple times to form random recombinant polynucleotides comprised of the ligated fragments;
wherein said method is performed in the absence of polymerase.
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Abstract
Ligation-mediated method of recombining polynucleotides in vitro. Polynucleotides from a library are fragmented and the fragments are hybridized to an assembly template. The hybridized fragments are iteratively re-hybridized and ligated until the ends of the hybridized fragments are adjacent to the ends of other hybridized fragments on the assembly template. A final ligation produces recombined polynucleotides.
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23 Claims
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1. A ligase-mediated method of in vitro recombination, comprising:
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providing oligonucleotide cleavage fragments derived from each of at least two different polynucleotides of a polynucleotide library;
hybridizing the cleavage fragments to an assembly template;
ligating together the hybridized fragments that have adjacent ends with a ligase;
denaturing the ligated fragments from the assembly template; and
repeating at least the hybridizing, ligating and denaturing steps multiple times to form random recombinant polynucleotides comprised of the ligated fragments;
wherein said method is performed in the absence of polymerase. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23)
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21. A ligation-mediated method of in vitro recombination, comprising:
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hybridizing, to an assembly template, fragments of polynucleotides derived from a polynucleotide library comprised of at least two different polynucleotides;
ligating together those hybridized fragments that have adjacent ends with a ligase;
denaturing the ligated fragments from the assembly template, and repeating the hybridizing, ligating and denaturing steps multiple times, thereby forming random recombinant polynucleotides comprised of the ligated fragments;
wherein the method is performed in the absence of a polymerase.
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22. A ligase-mediated method of in vitro recombination, comprising:
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providing oligonucleotide cleavage fragments derived from each of at least two different polynucleotides of a polynucleotide library;
hybridizing the cleavage fragments to an assembly template;
ligating together the hybridized fragments that have adjacent ends with a ligase;
denaturing the ligated fragments from the assembly template;
repeating at least the hybridizing, ligating and denaturing steps multiple times to form random recombinant polynucleotides comprised of the ligated fragments; and
selecting the formed recombinant polynucleotide that possesses advantageous properties compared to a reference sequence;
wherein said method is performed in the absence of polymerase.
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Specification