Multi-domain DNA ligands bound to a solid matrix for protein and nucleic acid affinity chromatography and processing of solid-phase DNA

• US 5,437,976 A
• Filed: 08/08/1991
• Issued: 08/01/1995
• Est. Priority Date: 08/08/1991
• Status: Expired due to Fees

• Current Assignee: Arizona Board of Regents (University of Arizona)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: Utermohlen, Joseph G.
• Primary Examiner(s): Walsh, Stephen G.
• Application Number:
  US07/742,856
• Time in Patent Office: 1,454 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.4, 935/19, 935/20, 935/21
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The present invention is directed to a multi-domain oligonucleotide ligand having multiple functions conferred by each of the domains. The ligand is bound to a solid phase matrix for use in both mRNA-affinity chromatography and as a priming matrix for generating matrix-bound cDNA from the mRNA bound to the matrix. The resultant cDNA was tested and found to function as a solid-phase template for PCR. This solid-phase template approach to PCR is demonstrated with oligo-d(T) paper. The functional domains are defined by polynucleotide sequences on the ligand to confer the different functions.

Claims

1. A molecular binding medium, comprising a solid matrix and an oligonucleotide ligand bound thereto, said oligonucleotide ligand comprising at least one nucleotide sequence defining a first functional domain and at least one nucleotide sequence defining a second functional domain, said first functional domain having a greater binding affinity for said solid matrix than said second functional domain;

wherein said first functional domain comprises a polynucleotide sequence defining a 5'"'"' terminal domain of said oligonucleotide ligand;

wherein said oligonucleotide is substantially directionally bound to said solid matrix at said 5'"'"' terminal domain thereof;

wherein said second functional domain is a 3'"'"' terminal domain of said oligonucleotide ligand;

wherein said first functional domain comprises a polynucleotide selected from the group consisting of polydeoxyguanosine of from about 4 to about 12 nucleic acid residues, polydeoxyuracil of from about 4 to about 12 nucleic acid residues, and poly-N-6-aminohexyldeoxyadenosine of from about 4 to about 12 nucleic acid residues; and

wherein said second functional domain comprises a polynucleotide comprising polydeoxythymidine of from about 18 to about 30 nucleic acid residues.

2. The binding medium of claim 1, wherein said solid matrix comprises an arylamine-substituted matrix.

3. The binding medium of claim 2, wherein said arylamine-substituted matrix is selected from the group consisting of arylamine-substituted cellulose, arylamine-substituted plastics, arylamine-substituted elastomers, arylamine-substituted resins, arylamine-substituted glucose polymers, arylamine-substituted polyacrylamides, arylamine-substituted agarose, arylamine-substituted rayon, arylamine-substituted nylon, arylamine-substituted triacetate, arylamine-substituted cellulose acetate, and arylamine-substituted cellulose nitrate.

4. The binding medium of claim 2, wherein said arylamine-substituted matrix is a cellulose paper selected from the group consisting of aminobenzyloxymethyl-derivatized cellulose paper and aminophenylthioether cellulose paper.

5. The binding medium of claim 1, further comprising a cDNA bound to said oligonucleotide ligand.

6. A DNA ligand, comprising a first polynucleotide sequence defining a first functional domain of the ligand and a second polynucleotide sequence defining a second functional domain of the ligand, wherein said first functional domain has a relatively greater binding affinity for a pre-selected solid matrix than said second functional domain;

wherein said first functional domain comprises a polynucleotide sequence defining a 5'"'"' terminal domain of said oligonucleotide ligand;

wherein said first functional domain comprises a polynucleotide selected from the group consisting of polydeoxyguanosine of from about 4 to about 12 nucleic acid residues, polydeoxyuracil of from about 4 to about 12 nucleic acid residues, and poly-N-6-aminohexyldeoxyadenosine of from about 4 to about 12 nucleic acid residues; and

wherein said second functional domain comprises a polynucleotide comprising polydeoxythymidine of from about 18 to about 30 nucleic acid residues.

7. The DNA ligand of claim 6, wherein said solid matrix comprises an arylamine-substituted matrix.

8. The DNA ligand of claim 6, wherein said arylamine-substituted matrix is selected from the group consisting of arylamine-substituted cellulose, arylamine-substituted plastics, arylamine-substituted elastomers, arylamine-substituted resins, arylamine-substituted glucose polymers, arylamine-substituted polyacrylamides, arylamine-substituted agarose, arylamine-substituted rayon, arylamine-substituted nylon, arylamine-substituted triacetate, arylamine-substituted cellulose acetate, and arylamine-substituted cellulose nitrate.

9. The DNA ligand of claim 6, wherein said arylamine-substituted matrix is a cellulose paper selected from the group consisting of aminobenzyloxymethyl-derivatized cellulose paper and aminophenylthioether cellulose paper.

10. The DNA ligand of claim 6, wherein said second functional domain comprises an active binding region separate from said first functional domain.

11. The DNA ligand of claim 6, wherein said second functional domain comprises a polynucleotide having a reactivity to arylamines which is less than the reactivity of at least one of deoxyguanosine or deoxyuracil to arylamines.

12. The DNA ligand of claim 6, further comprising a cDNA bound to said oligonucleotide ligand.

13. A matrix for mRNA affinity chromatography, comprising a substantially planar solid matrix having an oligonucleotide ligand bound thereto, said oligonucleotide ligand comprising a 5'"'"' domain having a greater binding affinity for said solid matrix than a 3'"'"' domain thereof and said oligonucleotide being substantially bound to said matrix by said 5'"'"' domain;

wherein said 5'"'"' domain comprises a first polynucleotide and said 3'"'"' domain comprises a second polynucleotide;

wherein said first polynucleotide is selected from the group consisting of polydeoxyguanosine of from about 4 to about 12 nucleic acid residues, polydeoxyuracil of from about 4 to about 12 nucleic acid residues, and poly-N-6-aminohexyldeoxyadenosine, of from about 4 to about 12 nucleic acid residues; and

wherein said second polynucleotide is polydeoxythymidine of from about 18 to about 30 nucleic acid residues.

14. The matrix of claim 13, further comprising a cDNA bound to said oligonucleotide ligand.

15. A matrix for detecting, amplifying and/or cloning nucleic acid sequences, comprising, in combination, a solid matrix and at least one multi-functional oligonucleotide ligand bound to said solid matrix, said oligonucleotide ligand comprising a 3'"'"' terminal domain polynucleotide and a 5'"'"' terminal domain polynucleotide, wherein said 5'"'"' terminal domain polynucleotide defines a region having greater binding affinity for said solid matrix and said 3'"'"' terminal domain polynucleotide defines a region for binding other polynucleotides and said oligonucleotide is substantially bound to said matrix by said 5'"'"' terminal domain;

wherein said 3'"'"' terminal domainpolynucleotide is polydeoxythymidine of from about 18 to about 30 nucleic acid residues, and said 5'"'"' terminal domain polynucleotide is selected from the group consisting of polydeoxyguanosine of from about 4 to about 12 nucleic acid residues and polydeoxyuracil of from about 4 to about 12 nucleic acid residues.

16. The matrix of claim 15, further comprising a RNA bound to said 3'"'"' terminal domain and a cDNA complement of said bound RNA, said cDNA being terminally bound to said 3'"'"' terminal domain polynucleotide.