Process for the production of expression vectors comprising at least one stochastic sequence of polynucleotides
DCFirst Claim
1. A process for the production of an expression vector which comprises at least one stochastic sequence of polynucleotides, comprising the steps of:
- providing in an appropriate buffer at least three different sequences of oligonucleotides, said oligonucleotides each comprising at least 7 nucleotide residues;
polymerizing said oligonucleotides to form a stochastic sequence of polynucleotides; and
ligating said stochastic sequence of polynucleotides into a linearized expression vector.
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Abstract
The present invention is directed to a process for the production of a peptide, polypeptide, or protein having a predetermined property. In accordance with one embodiment, the process begins by producing by way of synthetic polynucleotide coupling, stochastically generated polynucleotide sequences. A library of expression vectors containing such stochastically generated polynucleotide sequences is formed. Next, host cells containing the vectors are cultured so as to produce peptides, polypeptides, or proteins encoded by the stochastically generated polynucleotide sequences. Screening or selection is carried out on such host cells to identify a peptide, polypeptide, or protein produced by the host cells which has the predetermined property. The stochastically generated polynucleotide sequence which encodes the identified peptide, polypeptide, or protein is then isolated and used to produce the peptide, polypeptide, or protein having the predetermined property.
392 Citations
46 Claims
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1. A process for the production of an expression vector which comprises at least one stochastic sequence of polynucleotides, comprising the steps of:
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providing in an appropriate buffer at least three different sequences of oligonucleotides, said oligonucleotides each comprising at least 7 nucleotide residues; polymerizing said oligonucleotides to form a stochastic sequence of polynucleotides; and ligating said stochastic sequence of polynucleotides into a linearized expression vector. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 16)
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11. A process for the production of an expression vector capable of producing a transcription product or a translation product comprising at least one stochastic sequence of polynucleotides, comprising the steps of:
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linearizing an expression vector; reacting said linearized expression vector with terminal transferase enzyme in the presence of desired ratios of deoxynucleotide-triphosphates of guanine, cytosine, thymidine, and adenine to form a stochastic polynucleotide sequence at each 3'"'"' extremity of said linearized vector; hybridizing said stochastic polynucleotide sequence at a 3'"'"' extremity of said linearized vector; and synthesizing a second strand from said 3'"'"' ends of said hybridized vector by incubating with polymerase. - View Dependent Claims (15)
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12. A process for the production of a library of expression vectors capable of producing a transcription product or a translation product, said vectors comprising at least one stochastic sequence of polynucleotides, comprising the steps of:
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producing at least one stochastic sequence of polynucleotides; ligating said stochastic sequence of polynucleotides into an expression vector; transforming a competent clone with said ligated expression vector; culturing said transformed clone; screening and/or selecting said transformed clone in order to isolate a clone expressing a stochastic polynucleotide leading to the synthesis of a transcription product or a translation product; isolating said selected or screened transformed clone; and isolating the expression vector cultured in said selected or screened transformed clone so identified. - View Dependent Claims (13, 17)
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18. A method of producing a diverse population of vectors comprising:
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(a) synthesizing a diverse population of stochastically generated polynucleotide sequences comprising greater than about 1×
105 different polynucleotide sequences, said method consisting of stochastic copolymerization of double stranded oligonucleotides, copolymerization of the four kinds of nucleotide triphosphates consisting of adenine, cytosine, guaninie and thymine, and chemical synthesis, and(b) inserting said diverse population of stochastically generated polynucleotide sequences into a population of vectors to form a diverse population of vectors containing stochastically generated polynucleotide sequences. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26)
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27. A method of producing a diverse population of vectors, comprising stochastically copolymerizing a diverse population of vectors containing double stranded polynucleotides so as to produce a new population of vectors containing greater than about 1×
- 105 different polynucleotide sequences.
- View Dependent Claims (28, 29, 30, 31, 32, 33, 34, 35, 36)
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37. A method of producing a diverse populations of vectors, comprising:
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(a) obtaining one or more diverse populations of vectors containing diverse sequences of double stranded polynucleotides; (b) digesting the one or more diverse populations of vectors with a restriction enzyme, and (c) stochastically copolymerizing the one or more diverse populations of double stranded polynucleotides so as to produce a new population of greater than about 1×
105 different polynucleotide sequences. - View Dependent Claims (38, 39, 40, 41, 42, 43, 44, 45, 46)
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Specification