Patent ID: 8357638

Claim:
Method of producing an oligonucleotide library comprising a plurality of oligonucleotides, each oligonucleotide in the library having at least one predetermined position, a randomisation codon selected from a defined group of codons, the codons within said defined group coding for different amino acids, said method comprising the steps of: (a) Providing one or more double-stranded starter oligonucleotides, wherein the starter oligonucleotides comprise a blunt end; (b) Providing a plurality of different double stranded randomisation oligonucleotides comprising: (i) a coding strand, the coding strand comprising a randomisation codon; and (ii) a substantially complementary non-coding strand, wherein each double stranded randomisation oligonucleotide comprises a nucleotide sequence coding for a restriction endonuclease recognition site capable of being recognised by a restriction endonuclease, the restriction endonuclease capable of cleaving the randomisation oligonucleotide upstream or downstream of the endonuclease recognition site at a predetermined cleavage site to create a blunt ended cut; (c) Ligating each double-stranded starter oligonucleotide to a double-stranded randomisation oligonucleotide to form ligated oligonucleotides; (d) Amplifying the ligated oligonucleotides; (e) Digesting the ligated oligonucleotide with the restriction endonuclease to form a plurality of randomised double-stranded oligonucleotides, each of which comprise, at one end, a randomisation codon; and, (f) Using the randomised double-stranded oligonucleotides as starter oligonucleotides and repeating method steps (a) to (e) to produce a plurality of randomised double-stranded oligonucleotides, each comprising an additional randomisation codon; wherein step (f) is followed one or more times to produce an oligonucleotide library comprising a plurality of oligonucleotides, each oligonucleotide in the library having at least two contiguous randomised codons.