Cloning and copying on surfaces

• US 6,534,271 B2
• Filed: 05/25/2001
• Issued: 03/18/2003
• Est. Priority Date: 11/27/1998
• Status: Expired due to Term

• Current Assignee: NOXXON Pharma AG (TME Pharma NV)
• Original Assignee: NOXXON Pharma AG (TME Pharma NV)
• Inventors: Klein, Thomas, Von Kiedrowski, Günter, Fürste, Jens Peter, Klussmann, Sven
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/866,513
• Publication Number:
  US 20020022275A1
• Time in Patent Office: 662 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/7.1, 436/94
• US Class Current: 435/6.1
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: C12Q 2565/515 characterised by the interaction between or sequential use of two or more arrays
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification
• CPC Class Codes: Y10S 977/902 Specified use of nanostructure

Abstract

The present invention provides for amplification methods for cloning and copying genetic material on surfaces as well as copying biological material insofar as, in a broader sense, it can be classified as a ligand-receptor system. The invention therefore relates in particular to a method for propagating ligands and receptors on at least two surfaces, comprising (a) immobilizing a first ligand on a first surface of a substantially solid phase; (b) adding a solution of receptors and binding complementary receptors to the first ligand; (c) transferring the receptor to a second surface and immobilizing the receptor at that location; (d) attaching an additional ligand to the immobilized receptor; and (e) transferring the additional ligand to the first surface and immobilizing it at that location, wherein the steps set forth above may be repeated,multiple times.

Claims

1. A method for propagating ligands and receptors on at least two surfaces, comprising one or more of the following cycles:

(a) immobilizing a first ligand on a first surface of a solid phase;

(b) adding a solution of receptors and binding complementary receptors to the first ligand;

(c) transferring the receptor to a location on a second surface and immobilizing the receptor at that location;

(d) attaching an additional ligand to the immobilized receptor; and

(e) transferring the additional ligand to the first surface and immobilizing it at that location, wherein the steps set forth above may be repeated multiple times.

2. The method according to claim 1, wherein the transfer in stage (c) and (e) is achieved by the application of an electrical field.

3. The method according to claim 2, wherein the electrical field is applied between the first and second surface.

4. The method according to claim 1, wherein the solid phase is selected from a member of the group consisting of organic or inorganic material or from a hybrid of these materials, and represents a two- or three-dimensional matrix.

5. A method for strengthening an immunological ligand/receptor pair, comprising one or more of the following cycles:

(a) immobilizing a first ligand on a first surface of a solid phase;

(b) adding a solution of receptors and binding complementary receptors to the first ligand;

(c) transferring the receptor to a location on a second surface and immobilizing the receptor at that location;

(d) attaching an additional ligand to the immobilized receptor; and

(e) transferring the additional ligand to the first surface and immobilizing it at that location, thus strengthening an immunological ligand/receptor pair, wherein the steps set forth above may be repeated multiple times.

6. A method for strengthening a ligand signal, comprising one or more of the following cycles:

(a) immobilizing a first ligand on a first surface of a solid phase;

(b) adding a solution of receptors and binding complementary receptors to the first ligand;

(c) transferring the receptor to a location on a second surface and immobilizing the receptor at that location;

(d) attaching an additional ligand to the immobilized receptor; and

(e) transferring the additional ligand to the first surface and immobilizing it at that location, thus strengthening an ligand/signal, wherein the steps set forth above may be repeated multiple times.

7. A method for the enzymatic propagation of a nucleic acid sequence on at least two surfaces, comprising:

(a) immobilizing a first primer on at least one first surface of a solid phase;

(b) administering a solution of nucleic acids comprising complementary fragments to the first primer;

(c) binding of complementary fragments to the first primer;

(d) extending the first primer at its 3′

end, corresponding to the complementary fragment by means of a polymerase;

(e) releasing the complementary fragments;

(f) attaching a second primer to the 3′

end of the extended nucleic acid;

(g) extending the second primer at its 3′

end by means of a polymerase;

(h) transferring the second primer to another surface and immobilization of the extended primer; and

(i) attaching another first primer to the 3′

end of the second extended primer for further extending of the first primer.

8. The method according to claim 7, wherein the surface in step (h) is a second surface which is spatially separated from the first.

9. The method according to claim 7, wherein the transfer in step (h) is achieved by the application of an electrical field.

10. The method according to claim 9, wherein the electrical field is applied between the first and second surface.

11. The method according to claims 7, further comprising the following amplification steps

(j) extending this first primer to its 3′ end, corresponding to the complementary fragment, by means of a polymerase;

(k) transferring of the extended primer to the first or another surface and immobilization of the extended primer thereon; and

(l) attaching of another second primer to the 3′

end of the extended first primer.

13. The method according to claim 11, wherein the nucleic acids on the solid phase surface are arranged two-dimensionally and are transferred in this order, while retaining site information.

14. The method according to claim 11, wherein the solid phase surface is selected from a member of the group consisting of organic or inorganic material or from a hybrid of these materials, and represents a two- or three-dimensional matrix.

15. The method according to claims 11, wherein immobilization on the solid phase takes place through covalent or non-covalent binding.

16. The method according to claim 7, where the nucleic acids are provided with a detectable label.

17. The method according to claim 16, wherein the label is selected from a member of the group consisting of radioisotopes, stable isotopes, enzymes, immunoreactive compounds, fluorescence or luminescence chemicals, chromophores, metals or charged particles.

18. The method according to claim 7, wherein the nucleic acids include D- and/or L-nucleic acids.

19. The method according to claim 7 wherein an intermediate layer is placed between the surfaces.

20. The method according to claim 19, wherein the intermediate layer is selected from the group consisting of gel, a membrane, a polymer, a ceramic and capillary tube array.

21. The method according to claim 7, wherein the nucleic acids are each provided with a positive charge.

12. A method for cloning genomic fragments of DNA, cDNA and RNA, comprising:

(a) immobilizing a first primer on at least one first surface of a solid phase;

(b) administering a solution of nucleic acids comprising complementary fragments to the first primer;

(c) binding of complementary fragments to the first primer;

(d) extending the first primer at its 3′

end, corresponding to the complementary fragment by means of a polymerase;

(e) releasing the complementary fragments;

(f) attaching a second primer to the 3′

end of the extended nucleic acid;

(g) extending the second primer at its 3′

end by means of a polymerase;

(h) transferring the second primer to another surface and immobilization of the extended primer;

(i) attaching another first primer to the 3′

end of the second extended primer for further extending of the first primer;

(j) extending this first primer to its 3′

end, corresponding to the complementary fragment, by means of a polymerase;

(k) transferring of the extended primer to the first or another surface and immobilization of the extended primer and (m) cloning fragments produced by the above steps thereon;

(l) attaching of another second primer to the 3′

end of the extended first primer.

22. A method for sorting adjacent fragments by using hybridization techniques, comprising:

(a) immobilizing a first primer on at least one first surface of a solid phase;

(b) administering a solution of nucleic acids comprising complementary fragments to the first primer;

(c) binding of complementary fragments to the first primer;

(d) extending the first primer at its 3′

end, corresponding to the complementary fragment by means of a polymerase;

(e) releasing the complementary fragments;

(f) attaching a second primer to the 3′

end of the extended nucleic acid;

(g) extending the second primer at its 3′

end by means of a polymerase;

(h) transferring the second primer to another surface and immobilization of the extended primer;

(i) attaching another first primer to the 3′

end of the second extended primer for further extending of the first primer;

(j) extending this first primer to its 3′

end, corresponding to the complementary fragment, by means of a polymerase;

(k) transferring of the extended primer to the first or another surface and immobilization of the extended primer thereon;

(l) attaching of another second primer and (m) sorting adjacent fragments produced by the above steps to the 3′

end of the extended first primer.