Microcapsules with controlable permeability encapsulating a nucleic acid amplification reaction mixture and their use as reaction compartment for parallels reactions

• US 20050202429A1
• Filed: 03/20/2003
• Published: 09/15/2005
• Est. Priority Date: 03/20/2002
• Status: Abandoned Application

• Current Assignee: Innovativebio.Biz
• Original Assignee: Innovativebio.Biz
• Inventors: Renneberg, Reinhard, Trau, Dieter, Wing Cheung, Mak
• Application Number:
  US10/508,686
• Publication Number:
  US 20050202429A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 13/02 Making microcapsules or microballoons for medical preparations A61K9/50
• CPC Class Codes: B01J 13/14 Polymerisation; cross-linking
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

The present invention refers to microcapsules with high permeability for low molecular weight molecules and no permeability for high molecular weight molecules encapsulating a nucleic acid amplification reaction mixture, in particular, a PCR reaction mixture and providing a compartment to perform a nucleic acid amplification, in particular, a PCR reaction and to applications of such capsules to perform parallel PCR, screening, construction of DNA libraries and sequencing.

Claims

1. A nucleic acid amplification process comprising the steps

(a) providing a nucleic acid amplification reaction mixture,

(b) encapsulating said reaction mixture by a capsule being permeable for low molecular weight molecules and not permeable for high molecular weight molecules and thereby forming capsules comprising constituents of the reaction mixture, and

(c) performing at least one amplification or/and polymerization step and thereby amplifying a nucleic acid within one or more of the capsules.

2. The process according to claim 1, further comprising the step

(d) detecting capsules containing a nucleic acid product.

3. The process according to claim 1 or 2, further comprising the step

(e) separating capsules containing nucleic acid product from capsules not containing nucleic acid products.

4. The process according to any of the preceding claims, further comprising the step

(f) analyzing or extracting the nucleic acid product.

5. The process according to claim 1, wherein the constituents of the nucleic acid amplification reaction mixture are incorporated into a matrix material.

6. The process according to claim 5, wherein the matrix material is selected from a porous microparticle, a wax-like substance, an alginate, an agarose, a polysaccharide, a polypeptide, a fatty acid, a salt of a fatty acid, polyvinyl pyrrolidone (PVP), or the matrix is ice, or a mixture thereof.

7. The process according to any of the preceding claims, wherein the nucleic acid amplification reaction mixture incorporated into a matrix material is encapsulated using a layer-by-layer process, a polymerization process or/and a cavaciation process.

8. The process according to any of claims 5 to 7, wherein the matrix material is dissolved and/or removed from the capsule with solvents, by chemical means or by physical means such as heat.

9. The process according to claims 5 to 7, wherein the matrix material is melted during temperature cycling.

10. The process of any of the preceding claims, wherein capsules are formed having a diameter from 50 nm to 1000 μ

m, with a preferred diameter of 40 to 60 μ

m.

11. The process according to any of the preceding claims, wherein constituents and/or reactants of the nucleic acid amplification reaction mixture are transferred into the capsule by a triggered change in capsule wall permeability.

12. The process according to claim 11, wherein the change in capsule wall permeability is triggered by a pH change or an ionic strength change or a change in capsule cross linking.

13. A process according to claim 11 or 12, wherein the permeability of the capsule is controlled to allow selective diffusion of nucleic acid amplification reaction starting materials into the capsules but restrict the out-diffusion of nucleic acid amplification reaction products and/or templates and/or primers.

14. The process according to any of the preceding claims, wherein non-encapsulated nucleic acid amplification reaction mixture compounds are separated from the capsules or destructed by enzymes prior to the amplification step.

15. The process according to any of the preceding claims, wherein one or more components of the nucleic acid amplification reaction mixture and/or of the capsule material carries a label.

16. The process according to claim 15, wherein the label is selected from a fluorophore, a quantum dot, a radioisotope, a dye, a nanoparticle or an NMR active isotope.

17. The process according to claim 15 or 16, wherein the label enables the encoding of individual capsules.

18. The process according to any of the preceding claims, wherein capsules containing nucleic acid products are detected by the physical signature caused by a label or by UV adsorption measurement of DNA, or by dyes staining the DNA, and are separated and isolated from the capsule suspension.

19. The process according to any of the preceding claims for the analysis and/or screening of nucleic acids or artificial derivatives of nucleic acids.

20. The process according to any of the preceding claims for the production of a nucleic acid library.

21. The process of any of the preceding claims employed in a bioassay.

22. The process according to any of the preceding claims, characterized in that the nucleic acid amplification reaction is a polymerase chain reaction (PCR).

23. The process according to claim 22, comprising the steps

(i) providing a PCR reaction mixture consisting of polymerase and/or dNTPs and/or buffer substances and/or enhancers and/or primers and/or templates and/or DNA probes and/or matrix material,

(ii) encapsulating said PCR reaction mixture and thereby forming capsules comprising constituents of the reaction mixture and

(iii) performing at least one amplification step comprising PCR temperature cycling and thereby amplifying a nucleic acid within one or more of the capsules.

24. The process according to any of claims 11-13 or 22-23, wherein the template and/or one or more primers are transferred into the capsule by a triggered change in capsule wall permeability.

25. The process according to any of claims 11-13 or 22-24, wherein the permeability of the capsule is controlled to allow selective diffusion of dNTPs and/or ddNTPs and/or small primers into the capsules but restrict out-diffusion of polymerase, PCR products, templates and large primers during the temperature cycling.

26. The process according to any of claims 22-25, wherein the capsules are dispersed in a PCR buffer.

27. The process according to claim 26, wherein the PCR buffer contains intercalating dyes such as ethidium bromide and/or SYBR Green I.

28. The process according to any of claims 22-27, wherein the temperature cycling of the capsule suspension is carried out in reaction tubes, multititer plates or glass capillaries, using a thermocycler.

30. The process according to any of claims 15-16 or 29, wherein the label enables the encoding of primers and/or templates and/or mixtures thereof.

31. The process according to any of claims 22-30, wherein further enzymes such as reverse transcriptases and/or nucleases and/or ligases are added into the encapsulated PCR reaction mixture.

32. A method of sequencing the nucleic acid content of capsules obtained by any of claims 1-31 comprising the steps:

(i) performing the PCR amplification with an excess of one primer until the primer with the lower concentration is used Up.

(ii) adding 4 color fluorescent labeled ddNTPs at an amount of 1;

100 to 1;

1000 of dNTPs and elongating the excess primer and

(iii) analyzing the nucleic acid capsule content of individual capsules by gel electrophoresis.

33. A method of analyzing the nucleic acid content of capsules obtained by any of claims 1-31 comprising the steps:

(i) embedding capsules into an agarose or polyacrylamide gel,

(ii) releasing nucleic acid from the capsule by chemical or physical means.

(iii) applying an electrical potential to carry out a gel electrophoresis and

(vi) detecting the band patterns created from individual capsules.

34. Capsule encapsulating a nucleic acid amplification reaction mixture and/or a nucleic acid amplification product obtainable by any of claims 1-31.

35. Capsule according to claim 34, encapsulating a PCR reaction mixture and/or a PCR product.

36. Use of a capsule according to claim 34 or 35 for the analysis and/or screening of nucleic acids or artificial derivatives of nucleic acids, for the production of nucleic acid libraries, nucleic acid sequencing, in bioassays or/and in real-time PCR.

37. Use of the capsules according to claim 34 or 35 as reaction compartments for parallel nucleic acid amplification reactions.

38. Use according to claim 37 as reaction compartment for parallel PCR reactions.

39. A kit for the production of capsules according to any previous claim, and/or the performance of the process of any previous claim and/or the use of capsules according to any previous claim, containing polymerase and/or dNTPs and/or ddNTPs and/or buffer substances and/or enhancers and/or primers and/or templates and/or DNA probes and/or matrix material and/or capsule material and/or labels and/or plastic ware and/or a user manual.

29. The process according to any claims, wherein dNTPs and or ddNTPs and/or primers and/or templates and/or DNA probes and/or capsule material carry a label.