METHOD FOR THE SPATIAL ARRANGEMENT OF SAMPLE FRAGMENTS FOR AMPLIFICATION AND IMMOBILIZATION FOR FURTHER DERIVATIZATIONS

• US 20140228245A1
• Filed: 10/01/2012
• Published: 08/14/2014
• Est. Priority Date: 09/30/2011
• Status: Active Grant

• Current Assignee: Biocopy GmbH
• Original Assignee: Albert-Ludwigs-Universitaet Freiburg
• Inventors: Roth, Guenter, Hoffmann, Jochen
• Granted Patent: US 10,093,954 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00333 Closures attached to the reactor vessels
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2565/515 characterised by the interaction between or sequential use of two or more arrays
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The invention relates to a method for performing a biochemical or chemical reaction for an isolated, spatially separated amplification of sample fragments during a simultaneous immobilization and spatial arrangement of the sample fragments and reaction products, the amplification products, on one or more suitable solid phases for subsequent derivatizations.

Claims

1. A method for amplification of sample fragments, comprising:

a. supplying a solid phase with a microstructure, wherein the microstructure subdivides the solid phase so that at least one, preferably several compartments are formed, and wherein capture molecules are attached to the solid phase,

b. introducing at least one sample fragment into at least one compartment,

c. introducing a reaction mixture for amplification in at least one compartment,

d. separating and/or covering the individual compartments to obtain sealed compartments,

e. amplifying each sample fragment independently in the corresponding sealed compartment to obtain an amplification products,

f. wherein the amplification products generated by the amplifying in (e) are immobilized on the capture molecules.

2. The method according to claim 1, wherein the amplification products are analyzed, sequenced and/or derivatized.

3. The method according to claim 1, wherein the capture molecules are primers, preferably primers that are used for the amplifying in (e).

4. The method according to claim 1, wherein the amplification products are immobilized on the capture molecules via covalent bonds.

5. The method according to claim 1, wherein the compartments are cavities.

6. The method according to claim 1, wherein the introducing of the sample fragment and the introducing of the reaction mixture take place in one step, preferably by mixing the sample fragment with the reaction mixture before the introducing.

7. The method according to claim 1, wherein the reaction mixture and/or the sample fragment is/are added to the compartments by means of centrifugation, vacuum, atomization, immersion, dip coating and/or painting the surfaces of the solid phase.

8. The method according to claim 1, wherein the surfaces of the solid phase are made of semiconducting, amorphous, crystalline and/or fibrous materials.

9. The method according to claim 1, wherein the surfaces have hydrophilic and/or hydrophobic regions and/or are structured in one or more layers as a structured surface.

10. The method according to claim 1, wherein the covering and/or separating of the compartments include oil, electric forces, phase transitions, inert regions and/or covers.

11. The method according to claim 1, wherein the capture molecules have a spacer molecule and/or comprise a chemical coupling group at one or more ends.

12. The method according to claim 1, wherein the capture molecules preferably have a length of 1 to 1000 individual molecules.

13. The method according to claim 1, wherein the capture molecule are immobilized either directly or indirectly on the solid phase.

14. The method according to claim 1, wherein the cover has comprises capture molecules.

15. The method according to claim 1, wherein the capture molecules comprise at least one binding site for sample fragments and/or amplification products.

16. The method according to claim 1, wherein the capture molecules and/or amplification products have a photoactivatable cleavage group.

17. The method according to claim 1, wherein the capture molecules have a proton binding site for an RNA polymerase or a cell-free expression system.

18. The method according to claim 1, wherein the sample fragments are nucleic acids, preferably DNA or RNA, proteins, antibodies, synthetic molecules, organic molecules and/or natural substances or have been released from cells or natural substances introduced into the compartments.

19. The method according to claim 1, wherein the sample fragments are amplified directly or indirectly via an incorporated label from DNA or RNA.

20. The method according to claim 1, wherein the sample fragments are present having a length of 1 to 10¹⁰ individual molecules in single-stranded or double-stranded form.

21. The method according to claim 1, wherein the sample fragments have adapters on one or both ends, wherein the adapters have a uniform sequence with a length of 1 to 1000 individual molecules and/or have different functional units.

22. The method according to claim 1, wherein amplification in (e) is selected in particular from the group consisting of PCR, RPA, RCA, SDA, NASBA and LAMP.

23. The method according to claim 1, wherein the reaction mixture comprises reaction-enhancing components, preferably BSA, Tween and/or agarose.

24. The method according to claim 1, wherein said amplifying does not involve an emPCR.

25. A device comprising

g) a solid phase with at least one, preferably several compartments, wherein capture molecules are bound to the surface of the solid phase, preferably to the compartments and

h) a separation and/or cover.

26. The device according to claim 25, wherein capture molecules are bound to the separation and/or the cover.

27. A master array comprising

i) a solid phase with at least one, preferably several compartments, wherein capture molecules are bound to the surface of the solid phase, preferably to the compartments, and wherein

j) amplification products and/or derivatives of an amplification reaction are bound to the capture molecules.

28. The master array of claim 27, comprising a separation and/or cover, wherein capture molecules are bound to the separation and/or cover and amplification products and/or derivatives of an amplification reaction are bound to the capture molecules.

29. (canceled)

30. (canceled)

31. A kit for amplification of sample fragments and/or for producing an array, comprising

a solid phase with at least one, preferably several compartments, wherein capture molecules are bound to the surface of the solid phase, preferably to the compartments

separation and/or cover and

at least one reaction mixture.

32. The kit according to claim 31, wherein capture molecules are bound to the separation and/or the cover.