Method for preselecting recombinant clones containing a specific nucleic acid sequence and subsequent transformation with preselected clones

• US 5,484,702 A
• Filed: 01/27/1994
• Issued: 01/16/1996
• Est. Priority Date: 01/27/1994
• Status: Expired due to Term

• Current Assignee: The Research Foundation of State University of New York
• Original Assignee: The Research Foundation for The State University of New York (State University of New York)
• Inventors: Ludwig, Linda B.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): TRAN, PAUL
• Application Number:
  US08/189,155
• Time in Patent Office: 719 Days
• Field of Search: 435/6, 435/7.1, 435/7.2, 435/7.5, 435/18, 435/91.2, 935/78, 935/81
• US Class Current: 435/6.17
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6813 Hybridisation assays

Abstract

A method for preselecting a nucleic acid sequence of interest is provided. The method comprises immobilizing an affinity molecule to a solid support matrix, wherein the affinity molecule is specific for a hapten or target molecule; hybridization of a "hook" comprised of either an oligonucleotide (complementary to the sequence of interest) labeled with a hapten or target molecule to the target recombinant vector containing the nucleic acid sequence of interest; capturing of the vector-hook via the hapten or target molecule by the respective immobilized affinity molecule for which it has binding specificity; liberating the captured vector DNA by enzymatic digestion of either the hook, or of the immobilized affinity molecule; and adding and incubating competent host cells to promote the introduction of recombinant vector DNA into the competent host cells. Also disclosed are embodiments in which a) the hook is incorporated as part of the vector by hybridization to a denatured vector, followed by second strand synthesis to include the hook as part of the newly synthesized strand; and b) the hook is a protein that binds a genetic element within or adjacent to the nucleic acid sequence of interest, and the immobilized affinity molecule has binding specificity for the protein.

Claims

1. A method for preselecting a gene or nucleic acid sequence of interest from other DNA, wherein a portion of the nucleic acid sequence of the gene or nucleic acid sequence of interest is known, comprising the steps of:

(a) immobilizing affinity molecules to a solid support matrix, wherein the affinity molecules are specific for a hapten label or target molecule label;

(b) hybridizing DNA to be preselected with an oligonucleotide hook complementary to the known portion of the sequence of interest under denaturing and subsequent annealing conditions, said oligonucleotide hook having incorporated hapten label or target molecule label;

(c) contacting the DNA of step (b) with the immobilized affinity molecules under conditions which promote the specific binding between the affinity molecules and the hapten label or target molecule label of the oligonucleotide hook for which it has binding specificity;

(d) washing the support matrix to remove unbound DNA; and

(e) liberating any bound DNA from the solid support matrix by enzymatic digestion of a component selected from the group consisting of the labeled oligonucleotide hook, and the immobilized affinity molecule;

wherein liberated DNA represents the preselected gene or nucleic acid sequence of interest.

2. The method of claim 1, wherein the haphen label is biotin, and the affinity molecules are streptavidin.

3. The method of claim 1, wherein the target molecule label is digoxigenin and the affinity molecules are digoxigenin-specific antibody.

4. The method of claim 1, wherein the oligonucleotide hook is a species selected from the group consisting of RNA, and ssDNA, and asDNA.

5. The method of claim 1, wherein the gene or nucleic acid sequence of interest is contained within a recombinant DNA vector, and further comprising

introducing the liberated DNA, containing said recombinant vector, into a competent host cell.

6. The method of claim 1, wherein the immobilized affinity molecules are attached to solid support matrix by covalent or noncovalent means.

7. The method of claim 6, wherein solid support matrix is treated after attachment of the affinity molecules so as to prevent non-specific binding of added DNA to areas of the matrix not bound by affinity molecules.

8. A method for preselecting a gene or nucleic acid sequence of interest from other nucleic acids, wherein a portion of the nucleic acid sequence of the gene or nucleic acid sequence of interest contains a genetic element that binds specifically to a binding protein, comprising the steps of:

(a) immobilizing affinity molecules to a solid support matrix, wherein the affinity molecules have binding specificity for the binding protein which does not inhibit, or is not inhibited by, binding between the binding protein and the genetic element;

(b) incubating the nucleic acid sequence containing the genetic element with a binding protein known to specifically bind to said genetic element under incubation conditions that promote specific binding;

(c) contacting the nucleic acid sequence and binding protein mixture of step (b) with the immobilized affinity molecules under conditions which promote the specific binding between the affinity molecules and the binding protein for which it has binding specificity;

(d) washing the support matrix to remove unbound nucleic acid sequences; and

(e) liberating any nucleic acid sequence bound to the binding protein by dissociation, or by enzymatic digestion of a component selected from the group consisting of the binding protein, and the immobilized affinity molecule, or a combination of both;

wherein liberated nucleic acid sequence represents the preselected gene or nucleic acid sequence of interest containing the genetic element.

9. The method of claim 8, wherein the genetic element is related to or contiguous with the gene or nucleic acid sequence of interest.

10. The method of claim 8, wherein the genetic element is an element bound by a protein selected from the group consisting of a DNA-binding protein and a RNA-binding protein.

11. The method of claim 8, wherein the affinity molecules are selected from the group consisting of polyclonal antibodies and monoclonal antibody, or a combination thereof.

12. The method of claim 8, wherein the immobilized affinity molecules are attached to solid support matrix by covalent or noncovalent means.

13. The method of claim 8, wherein solid support matrix is treated after attachment of the affinity molecules so as to prevent non-specific binding of added nucleic acid sequences to areas of the matrix not bound by affinity molecules.

14. The method of claim 8, wherein the gene or nucleic acid sequence of interest is contained within a recombinant DNA vector, and further comprising

introducing liberated DNA, containing said recombinant vector, into a competent host cell.

15. A method for preselecting a gene or nucleic acid sequence of interest from other DNA, wherein a portion of the nucleic acid sequence of the gene or nucleic acid sequence of interest is known, comprising the steps of:

(a) immobilizing affinity molecules to support matrix, wherein the affinity molecules are specific for a hapten label or target molecule label;

(b) denaturing DNA to be preselected and annealing the denatured DNA with a single-stranded oligonucleotide hook complementary to the known portion of the sequence of interest, said oligonucleotide hook having incorporated hapten label or target molecule label;

(c) synthesizing a second strand of the DNA containing the sequence of interest by an extension reaction, from the annealed labeled hook, containing a DNA polymerase which lacks exonuclease activity, wherein the labeled hook becomes incorporated as part of the synthesized double-stranded DNA;

(d) removing unannealed hook from labeled, synthesized double-stranded DNA of step (c);

(e) contacting the labeled, synthesized double-stranded DNA of step (d) with the immobilized affinity molecules under conditions which promote the specific binding between the affinity molecules and the hapten label or target molecule label in the synthesized DNA for which it has binding specificity;

(f) washing support matrix to remove unbound DNA; and

(g) liberating any bound DNA from solid support matrix by enzymatic digestion, or chemical dissociation of a component selected from the group consisting of the labeled oligonucleotide, and the immobilized affinity molecule;

wherein liberated DNA represent the preselected gene or nucleic acid sequence of interest.

16. The method of claim 15, wherein the hapten label is biotin, and the affinity molecules are streptavidin.

17. The method of claim 15, wherein the target molecule label is digoxigenin and the affinity molecules are digoxigenin-specific antibody.

18. The method of claim 15, wherein the oligonucleotide hook is a species selected from the group consisting of RNA, and ssDNA, and dsDNA.

19. The method of claim 15, wherein the gene or nucleic acid sequence of interest is contained within a recombinant DNA vector, and further comprising a step selected from the group consisting of

(a) introducing liberated DNA, containing said recombinant vector, into a competent host cell;

and

(b) further analyzing the sequence of the gene or nucleic acid of interest contained in the liberated DNA.

20. The method of claim 15, wherein the immobilized affinity molecules are attached to solid support matrix by covalent or noncovalent means.

21. The method of claim 20, wherein solid support matrix is treated after attachment of the affinity molecules so as to prevent non-specific binding of added DNA to areas of the matrix not bound by affinity molecules.