US5824469A
Expired / Lapsed
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Method for producing novel DNA sequences with biological activity
Assignee: WASHINGTON, University of
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Filing: 1994-09-30
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Granted: 1998-10-20
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Expires: 2014-09-30
Abstract
Abstract A method of obtaining an oligonucleotide capable of carrying out a predetermined biological function. A heterogeneous pool of oligonucleotides, x+y+z nucleotides in length, is first generated. Each oligonucleotide has a 5' randomized sequence, x nucleotides in length, a central preselected sequence, y nucleotides in length, and a 3' randomized sequence, z nucleotides in length. The resulting heterogeneous pool contains nucleic acid sequences representing a random sampling of the 4x+z possible sequences for oligonucleotides of the stated length. A random sampling of the heterogeneous pool of oligonucleotides is introduced into a population of cells that do not exhibit the predetermined biological function. The population of engineered cells is then screened for a subpopulation of cells exhibiting the predetermined biological function. From that subpopulation of cells is isolated an oligonucleotide containing the preselected sequence and capable of carrying out the predetermined biological function.
Inventors
- 👤 Marshall S. Horwitz
- 👤 Lawrence A. Loeb
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 00:57
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/66; C12N15/10; C12N15/102; C12N15/11; C12N15/62; C12N15/625; C12N15/70; C12N15/74; C12N15/79; C07; C07K; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/61
Claims Analysis
Total Claims: 6
Claim 1
Independent Claim
192 words
A method of obtaining an oligonucleotide that confers a predetermined biological function on a cell, comprising: (a) cloning into an expression vector a heterogeneous pool of oligonucleotides, x+y+z nucleotides in length, said oligonucleotides comprising a 5' randomized sequence x nucleotides in length, a central sequence y nucleotides in length, and a 3' randomized sequence z nucleotides in length, said heterogeneous pool having nucleic acid sequences representing a random sampling of the 4 x+z possible sequences for oligonucleotides of said length, wherein said cloned oligonucleotides are transcribed or act as a regulatory sequence: (b) introducing a random sampling of said cloned heterogeneous pool of oligonucleotides into a population of cells that do not exhibit the predetermined biological function, (c) thereafter biologically selecting said population of cells for a subpopulation of cells exhibiting said predetermined biological function by contacting the population of cells with a selective medium in which only cells exhibiting the predetermined biological function survive, and (d) isolating from said subpopulation of cells an oligonucleotide comprising said central sequence and that confers said predetermined biological function; wherein said predetermined biological function is regulation of expression or a biological activity of a polypeptide.
Claim 2
Independent Claim
192 words
A method of obtaining an oligonucleotide that confers a predetermined biological function on a cell, comprising: (a) cloning into an expression vector a heterogeneous pool of oligonucleotides, x+y+z nucleotides in length, said oligonucleotides comprising a 5' randomized sequence x nucleotides in length, a central sequence y nucleotides in length, and a 3' randomized sequence z nucleotides in length, said heterogeneous pool having nucleic acid sequences representing a random sampling of the 4 x+z possible sequences for oligonucleotides of said length, wherein said cloned oligonucleotides are transcribed or act as a regulatory sequence: (b) introducing a random sampling of said cloned heterogeneous pool of oligonucleotides into a population of cells that do not exhibit the predetermined biological function, (c) thereafter biologically selecting said population of cells for a subpopulation of cells exhibiting said predetermined biological function by contacting the population of cells with a selective medium in which only cells exhibiting the predetermined biological function survive, and (d) isolating from said subpopulation of cells an oligonucleotide comprising said central sequence and that confers said predetermined biological function; wherein said predetermined biological function is regulation of expression or a biological activity of a polypeptide.
Claim 3
Independent Claim
194 words
A method of obtaining an oligonucleotide that confers a predetermined biological function on a cell, comprising: (a) cloning into an expression vector a heterogeneous pool of oligonucleotides, n nucleotides in length wherein n is 54 or fewer nucleotides, from a mixture of nucleotides consisting essentially of a% adenine, t% thymidine, c% cytosine, and g% guanine, wherein a+t+c+g=100%, said heterogeneous pool having nucleic acid sequences representing a random sampling of the 4 n possible sequences for oligonucleotides of said length generated from nucleotides of said relative percent concentrations, wherein said cloned oligonucleotides are transcribed or act as a regulatory sequence; (b) introducing a random sampling of said cloned heterogeneous pool of oligonucleotides into a population of cells that do not exhibit the predetermined biological function; (c) thereafter biologically selecting said population of cells for a subpopulation of cells exhibiting said predetermined biological function, by contacting the population of cells with a selective medium in which only cells exhibiting the predetermined biological function survive; and (d) isolating from said subpopulation of cells an oligonucleotide that confers said predetermined biological function; wherein said predetermined biological function is regulation of expression or a biological activity of a polypeptide.
Claim 4
Independent Claim
194 words
A method of obtaining an oligonucleotide that confers a predetermined biological function on a cell, comprising: (a) cloning into an expression vector a heterogeneous pool of oligonucleotides, n nucleotides in length wherein n is 54 or fewer nucleotides, from a mixture of nucleotides consisting essentially of a% adenine, t% thymidine, c% cytosine, and g% guanine, wherein a+t+c+g=100%, said heterogeneous pool having nucleic acid sequences representing a random sampling of the 4 n possible sequences for oligonucleotides of said length generated from nucleotides of said relative percent concentrations, wherein said cloned oligonucleotides are transcribed or act as a regulatory sequence; (b) introducing a random sampling of said cloned heterogeneous pool of oligonucleotides into a population of cells that do not exhibit the predetermined biological function; (c) thereafter biologically selecting said population of cells for a subpopulation of cells exhibiting said predetermined biological function, by contacting the population of cells with a selective medium in which only cells exhibiting the predetermined biological function survive; and (d) isolating from said subpopulation of cells an oligonucleotide that confers said predetermined biological function; wherein said predetermined biological function is regulation of expression or a biological activity of a polypeptide.
Claim 5
Independent Claim
165 words
A method of obtaining an oligonucleotide that confers a predetermined biological function on a cell, comprising: (a) cloning into an expression vector a heterogeneous pool of oligonucleotides, n nucleotides in length wherein n is 54 or fewer nucleotides, having nucleic acid sequences representing a random sampling of the 4 n possible sequences for oligonucleotides of said length, wherein said cloned oligonucleotides are transcribed or act as a regulatory sequence; (b) introducing a random sampling of said cloned heterogeneous pool of oligonucleotides into a population of cells that do not exhibit the predetermined biological function; (c) thereafter biologically selecting from the population of cells a subpopulation of cells exhibiting the predetermined biological function, by contacting the population of cells with a selective medium in which only cells exhibiting the predetermined biological function survive; and (d) isolating from the selected subpopulation of cells an oligonucleotide that confers the predetermined biological function; wherein said predetermined biological function is regulation of expression or a biological activity of a polypeptide.
Claim 6
Independent Claim
165 words
A method of obtaining an oligonucleotide that confers a predetermined biological function on a cell, comprising: (a) cloning into an expression vector a heterogeneous pool of oligonucleotides, n nucleotides in length wherein n is 54 or fewer nucleotides, having nucleic acid sequences representing a random sampling of the 4 n possible sequences for oligonucleotides of said length, wherein said cloned oligonucleotides are transcribed or act as a regulatory sequence; (b) introducing a random sampling of said cloned heterogeneous pool of oligonucleotides into a population of cells that do not exhibit the predetermined biological function; (c) thereafter biologically selecting from the population of cells a subpopulation of cells exhibiting the predetermined biological function, by contacting the population of cells with a selective medium in which only cells exhibiting the predetermined biological function survive; and (d) isolating from the selected subpopulation of cells an oligonucleotide that confers the predetermined biological function; wherein said predetermined biological function is regulation of expression or a biological activity of a polypeptide.
References Cited (Backward Citations)
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