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US5824514A
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Process for the production of expression vectors comprising at least one stochastic sequence of polynucleotides
Assignee: Individual
•
Filing: 1995-06-05
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Granted: 1998-10-20
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Expires: 2015-06-05
Abstract
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.
Inventors
- 👤 Stuart Alan Kauffman
- 👤 Marc Ballivet
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 09:36
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/1093; C07; C07K; C07K14/00; C07K14/005; C07K14/435; C07K14/475; C07K14/485; C12N15/1048; C12N15/63; C12N15/64; C12N15/66; C12N15/67; C12N15/68; C12N15/70; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12N9/2468; C12N9/2471; C12Y; C12Y302/00; C12Y302/01; C12Y302/01023; A; A61; A61K; A61K39/00; C07K2319/00; C12N2730/00; C12N2730/00011; C12N2730/10011; C12N2730/10111; C12N2730/10122
Claims Analysis
Total Claims: 52
Claim 1
Independent Claim
65 words
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.
Claim 2
Dependent Claim
65 words
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.
Claim 3
Dependent Claim
13 words
The process according to claim 1 wherein the oligonucleotides further comprise a heptamer.
Claim 4
Dependent Claim
118 words
The process according to claim 1, said process further comprising the steps of: transforming a competent clone with said ligated expression vector; culturing said transformed competent clone; purifying said expression vector from said amplified competent clone; isolating said cultured stochastic sequence of polynucleotides from said expression vector; cutting said stochastic sequence of polynucleotides by means of at least one restriction enzyme which corresponds to a specific restriction enzyme site present in said stochastic sequence of polynucleotides; treating said cut stochastic sequence of polynucleotides with a T4 ligase to create a new ensemble of stochastic sequence of polynucleotides containing a new stochastic sequence of polynucleotides; and ligating said new ensemble of stochastic sequence of polynucleotides into an expression vector.
Claim 5
Dependent Claim
11 words
The process according to claim 2 wherein the heptamers are palindromic.
Claim 6
Dependent Claim
37 words
The process according to claim 4 wherein the palindromic heptamers are selected from the group consisting of: 5' XTCGCGA 3'; 5' XCTGCAG 3'; and 5' RGGTACC 3'; where X=A, G, C, or T, and R=A or T.
Claim 7
Dependent Claim
13 words
The process according to claim 1 wherein the oligonucleotides further comprise an octomer.
Claim 8
Dependent Claim
111 words
The process according to claims 1 or 6, said process further comprising the steps of: transforming a competent clone with said ligated expression vector; culturing said transformed competent clone; purifying and isolating said expression vector from said cultured competent clone; cutting said stochastic sequence of polynucleotides by means of at least one restriction enzyme which corresponds to said specific restriction enzyme site present in said stochastic sequence of polynucleotides; treating said cut stochastic sequence of polynucleotides with a T4 ligase to create a new ensemble of stochastic sequence of polynucleotides comprising a new stochastic sequence of polynucleotides; and ligating said new ensemble of stochastic sequence of polynucleotides into an expression vector.
Claim 9
Dependent Claim
11 words
The process according to claim 6 wherein the octamers are palindromic.
Claim 10
Dependent Claim
33 words
The process according to claim 8 wherein the palindromic octamers are selected from the group consisting of: 5' GGAATTCC 3'; 5' GGTCGACC 3'; 5' CAAGCTTG 3'; 5' CCATATGG 3'; and 5' CATCGATG 3'.
Claim 11
Dependent Claim
22 words
The process according to claim 1 wherein the stochastic double stranded DNA fragments are about 160 to 800 base pairs in length.
Claim 12
Dependent Claim
102 words
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: 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.
Claim 13
Dependent Claim
102 words
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: 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.
Claim 14
Dependent Claim
116 words
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: 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.
Claim 15
Dependent Claim
116 words
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: 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.
Claim 16
Dependent Claim
35 words
A library of expression vectors capable of producing a transcription product or a translation product, said expression vectors comprising at least one stochastic sequence of polynucleotides, produced in accordance with the process of claim 12.
Claim 17
Dependent Claim
14 words
An expression vector produced in accordance with the process of claim 1, 11, 12.
Claim 18
Dependent Claim
34 words
The process according to claims 11 or 12 wherein said translation product comprises a product having a desired property and is selected from the group consisting of a peptide, a polypeptide or a protein.
Claim 19
Dependent Claim
32 words
The process according to claims 1 or 12 wherein said transcription product comprises a product having a desired property and is selected from the group consisting of a RNA or a DNA.
Claim 20
Dependent Claim
25 words
The library of expression vectors according to claim 13 wherein said library comprises stochastic nucleotide sequences encoding for at least 10,000 peptides, polypeptides or proteins.
Claim 21
Independent Claim
85 words
A method of producing a diverse population of vectors comprising: (a) synthesizing a diverse population of stochastically generated polynucleotide sequences comprising greater than about 1×10 5 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.
Claim 22
Independent Claim
85 words
A method of producing a diverse population of vectors comprising: (a) synthesizing a diverse population of stochastically generated polynucleotide sequences comprising greater than about 1×10 5 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.
Claim 23
Dependent Claim
24 words
The method of claim 18, which said diverse population of stochastically generated polynucleotide sequences, further comprise greater than about 1×10 6 different polynucleotide sequences.
Claim 24
Dependent Claim
24 words
The method of claim 18, which said diverse population of stochastically generated polynucleotide sequences, further comprise greater than about 1×10 7 different polynucleotide sequences.
Claim 25
Dependent Claim
24 words
The method of claim 18, wherein said diverse population of stochastically generated polynucleotide sequences, further comprise greater than about 1×10 8 different polynucleotide sequences.
Claim 26
Dependent Claim
14 words
The method of claim 18, wherein said inserting further comprises hybridization of complementary ends.
Claim 27
Dependent Claim
11 words
The method of claim 18, wherein said inserting further comprises ligation.
Claim 28
Dependent Claim
21 words
The method of claim 18, further comprising introducing said diverse population of vectors containing stochastically generated polynucleotide sequences into host cells.
Claim 29
Dependent Claim
20 words
The method of claim 18, wherein step (a) further comprises synthesizing a population of at least partially stochastic polynucleotide sequences.
Claim 30
Dependent Claim
53 words
The method of claim 18, wherein step (b) further comprises digesting the diverse population of vectors with a restriction enzyme having a recognition sequence absent in the expression vector and reinserting the digested products into said digested population of vectors to form a different population having a greater number of stochastic polynucleotide sequences.
Claim 31
Independent Claim
39 words
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 1×10 5 different polynucleotide sequences.
Claim 32
Independent Claim
39 words
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 1×10 5 different polynucleotide sequences.
Claim 33
Dependent Claim
21 words
The method of claim 27, wherein said new population of vectors further comprise greater than about 1×10 6 different polynucleotide sequences.
Claim 34
Dependent Claim
21 words
The method of claim 27, which said new population of vectors further comprise greater than about 1×10 7 different polynucleotide sequences.
Claim 35
Dependent Claim
21 words
The method of claim 27, wherein said new population of vectors further comprise greater than about 1×10 8 different polynucleotide sequences.
Claim 36
Dependent Claim
16 words
The method of claim 27, wherein said stochastic copolymerization is effected by hybridization of complementary ends.
Claim 37
Dependent Claim
13 words
The method of claim 27, wherein said stochastic copolymerization is effected by ligation.
Claim 38
Dependent Claim
21 words
The method of claim 27, further comprising introducing said diverse population of vectors containing stochastically generated polynucleotide sequences into host cells.
Claim 39
Dependent Claim
15 words
The method of claim 27, wherein said double stranded polynucleotides further comprise stochastic polynucleotide sequences.
Claim 40
Dependent Claim
18 words
The method of claim 27, wherein said double stranded polynucleotides further comprise at least partially stochastic polynucleotide sequences.
Claim 41
Dependent Claim
20 words
The method of claim 27, wherein said diverse population of vectors further comprises two or more diverse populations of vectors.
Claim 42
Independent Claim
70 words
A method of producing a diverse populations of vectors, comprising: (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 1×10 5 different polynucleotide sequences.
Claim 43
Independent Claim
70 words
A method of producing a diverse populations of vectors, comprising: (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 1×10 5 different polynucleotide sequences.
Claim 44
Dependent Claim
21 words
The method of claim 37, wherein said new population of vectors further comprise greater than about 1×10 6 different polynucleotide sequences.
Claim 45
Dependent Claim
21 words
The method of claim 37, wherein said new population of vectors further comprise greater than about 1×10 7 different polynucleotide sequences.
Claim 46
Dependent Claim
21 words
The method of claim 37, wherein said new population of vectors further comprise greater than about 1×10 8 different polynucleotide sequences.
Claim 47
Dependent Claim
16 words
The method of claim 37, wherein said stochastic copolymerization is effected by hybridization of complementary ends.
Claim 48
Dependent Claim
13 words
The method of claim 37, wherein said stochastic copolymerization is effected by ligation.
Claim 49
Dependent Claim
21 words
The method of claim 37, further comprising introducing said diverse population of vectors containing stochastically generated polynucleotide sequences into host cells.
Claim 50
Dependent Claim
15 words
The method of claim 37, wherein said double stranded polynucleotides further comprise stochastic polynucleotide sequences.
Claim 51
Dependent Claim
18 words
The method of claim 37, wherein said double stranded polynucleotides further comprise at least partially stochastic polynucleotide sequences.
Claim 52
Dependent Claim
20 words
The method of claim 37, wherein said diverse population of vectors further comprises two or more diverse populations of vectors.
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