METHODS FOR CREATING ANTIBODY LIBRARIES
First Claim
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1. A method of preparing a library of vectors encoding different antibody sequences, comprising the steps of:
- a) annealing a first population of polynucleotides with at least a second population of polynucleotides, said first population comprising nucleotide sequences encoding an immunoglobulin complementarity determining region (CDR), wherein said CDR is diversified at one or more amino acid positions, said at least a second population comprising nucleotide sequences complementary to the nucleotide sequences comprised in said first population;
b) preparing double-stranded polynucleotides comprising nucleotide sequences encoding an immunoglobulin variable domain that incorporates the diversified CDR from extending strands of the annealed polynucleotides; and
c) inserting the double-stranded polynucleotides into a vector to provide a library of vectors, with two or more members of said library comprising different antibody coding sequences,wherein steps a), b) and c) are performed without the use of PCR amplification.
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Abstract
Methods and composition for the preparation of gene libraries of antibodies or parts of antibodies which contain the variable domains. For example, in certain aspects methods for providing polynucleotide library involving annealing and extending are described. Furthermore, the invention provides polynucleotide or antibody fragment libraries prepared by the methods.
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Citations
27 Claims
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1. A method of preparing a library of vectors encoding different antibody sequences, comprising the steps of:
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a) annealing a first population of polynucleotides with at least a second population of polynucleotides, said first population comprising nucleotide sequences encoding an immunoglobulin complementarity determining region (CDR), wherein said CDR is diversified at one or more amino acid positions, said at least a second population comprising nucleotide sequences complementary to the nucleotide sequences comprised in said first population; b) preparing double-stranded polynucleotides comprising nucleotide sequences encoding an immunoglobulin variable domain that incorporates the diversified CDR from extending strands of the annealed polynucleotides; and c) inserting the double-stranded polynucleotides into a vector to provide a library of vectors, with two or more members of said library comprising different antibody coding sequences, wherein steps a), b) and c) are performed without the use of PCR amplification. - View Dependent Claims (2, 3, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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4. A method of preparing a library of double-stranded polynucleotides, comprising the steps of:
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a) providing a first population of polynucleotides, said first population comprising nucleotide sequences encoding an immunoglobulin complementarity determining region (CDR), wherein said CDR is diversified at one or more amino acid positions; b) providing at least a second population of polynucleotides, said at least a second population comprising nucleotide sequences complementary to the nucleotide sequences comprised in said first population and capable of annealing to the first population of polynucleotides to form annealed polynucleotides that comprise nucleotide sequences encoding an immunoglobulin variable domain, wherein the immunoglobulin variable domain comprises the diversified CDR and an additional CDR; c) annealing the polynucleotides of the first population and the polynucleotides of the at least a second population; and d) extending strands of the annealed polynucleotides to prepare a library of double-stranded polynucleotides. - View Dependent Claims (5, 6, 7, 8, 26, 27)
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Specification