Method for amplification of nucleic acids in solid media

• US 5,616,478 A
• Filed: 10/26/1992
• Issued: 04/01/1997
• Est. Priority Date: 10/14/1992
• Status: Expired due to Term

• Current Assignee: Institut Belka
• Original Assignee: Institut Belka
• Inventors: Chetverin, Alexander B., Chetverina, Helena V.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): CAMPBELL, EGGERTON A
• Application Number:
  US07/966,713
• Time in Patent Office: 1,618 Days
• Field of Search: 435/91.1, 435/91.21, 435/299, 435/6, 210/490, 210/500
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2547/107 Use of permeable barriers, e.g. waxes

Abstract

Amplification and/or expression of nucleic acids is carried out in a medium immobilized by using an organic and/or inorganic solid matrix penetrating the medium and having a porous, fibrous, reticulated, coiled, capillary, lamellar or folded texture and which includes the components of a cell-free enzyme system of exponential amplification of nucleic acids and/or components of a cell-free enzyme system of nucleic acid expression. In this medium, the progeny of each molecule (clone) and the expression products remain in the same zone of the reaction volume where the matrix molecule was initially located. The method permits cloning of nucleic acids in vitro as well as detection of solitary nuleic acid molecules in the sample studied.

Claims

1. A method of exponential nucleic acid amplification to form detectable colonies of nucleic acids comprising the steps of

(a) providing an immobilized medium, said medium including

(i) an aqueous liquid phase that includes a cell-free, enzymatic, exponential nucleic acid amplification system;

and

(i) a solid, water-insoluble matrix having an average pore size ranging from 100 μ

m to 5 nm, completely entrapping said liquid phase, and

(b) distributing in said aqueous liquid phase nucleic acid molecules, at least one of which may comprise a template for said amplification system; and

(c) incubating said immobilized medium containing said distributed molecules under conditions promoting synthesis of an exponentially amplified nucleic acid product by said amplification system from said at least one template,

wherein said matrix is stable under said conditions, and wherein said step of distributing separates individual templates, resulting in nucleic acid amplification to form at least one separate, detectable colony of said nucleic acid product in said medium.

2. The method according to claim 1 wherein the steps (a) and (b) are carried out under conditions suppressing amplification of nucleic acids.

3. The method according to claim 1 wherein the immobilized medium is shaped into at least one layer.

4. The method according to claim 1 wherein said amplification system is a Viral RNA Polymerase system.

5. The method according to claim 1 wherein said amplification system is a 3SR Amplification system.

6. The method according to claim 1 wherein said amplification system is a Polymerase Chain Reaction system and wherein said step (c) of incubating includes cycling the medium through at least two temperatures.

7. The method according to claim 1 wherein said solid matrix is selected from the group consisting of agarose, polyacrylamide, nylon, gelatin, alginate, carrageenan, cellulose, silica gel, titanium sponge, dextran, and polyethylene glycol.

8. The method according to claim 1 wherein said matrix is chemically modified with hydrophobic and positively charged groups.

9. The method according to claim 1 wherein said matrix is chemically modified with weak cationic groups selected from the group consisting of secondary and tertiary amino groups.

10. The method according to claim 1 wherein said matrix is modified with an intercalating dye.

11. The method according to claim 1 wherein said step (a) of providing an immobilized medium comprises distributing said aqueous liquid phase in said solid matrix.

12. The method according to claim 1 wherein said amplification system comprises nucleotide substrates selected from the group consisting of ribonucleotides, deoxyribonucleotides and both ribonucleotides and deoxyribonucleotides.

13. The method according to claim 12 wherein at least one of said nucleotide substrates is labeled.

14. A method of exponential nucleic acid amplification to form detectable colonies of nucleic acids utilizing a cell-free, enzymatic, exponential nucleic acid amplification process that comprises repeated temperature cycling, comprising the steps of

(a) mixing with a gel-producing solution a nucleic acid amplification system for said amplification process,

(b) casting from said gel-producing solution a first solid, water-insoluble gel matrix layer having an average pore size ranging from 5 nm to 100 μ

m, said matrix completely entrapping said amplification system,

(c) distributing in said amplification system nucleic acid molecules, at least one of which may comprise a template for said amplification system, and

(d) cycling the temperature of said first matrix layer repeatedly according to said amplification process,

wherein said first matrix layer is stable at said cycling temperatures and wherein said step of distributing separates individual templates, resulting in nucleic acid amplification to form at least one separate, detectable colony of said nucleic acid product in said first matrix layer.

15. The method according to claim 14 wherein said first matrix layer has a thickness of 50 μ

m to 10 mm.

16. The method according to claim 15 wherein said amplification process is a Polymerase Chain Reaction process.

17. The method according to claim 14 wherein during said step (d) of temperature cycling, said first matrix layer is contacted with at least one blotting membrane, and amplified product transfers to said at least one blotting membrane.

18. The method according to claim 17 wherein said amplification process is a Polymerase Chain Reaction process.

19. The method according to claim 14 wherein during said step (d) of temperature cycling, said first matrix layer is contacted with a second matrix layer having an average pore size ranging from 5 nm to 100 μ

m and containing at least one component of said amplification system, wherein said second matrix layer is stable at said cycling temperatures.

20. The method according to claim 14 wherein said step (c) of distributing nucleic acid molecules in the amplification system precedes said step (b) of casting the matrix from the solution containing the amplification system.

21. A method of exponential nucleic acid amplification to form detectable colonies of nucleic acids comprising the steps of

(a) providing a first solid, water-insoluble matrix layer having an average pore size ranging from 100 μ

m to 5 nm and containing a first portion of the components of a cell-free, enzymatic, exponential nucleic acid amplification system and having distributed therein nucleic acid molecules, at least one of which may comprise a template for said amplification system;

(b) providing a second, solid, water-insoluble matrix layer having an average pore size ranging from 100 μ

m to 5 nm and containing a second portion of the components of said amplification system, said first and second portions together comprising said amplification system;

(c) contacting said first and second matrix layers; and

(d) incubating said first and second matrix layers while maintaining contact therebetween under conditions promoting synthesis of an exponentially amplified nucleic acid product by said amplification system from said at least one template,

wherein said first and second matrix layers are stable under said conditions, wherein said conditions cause at least said first portion or said second portion of said amplification system to diffuse from one of said matrix layers into the other, and wherein said distribution of nucleic acid molecules separates individual templates, resulting in nucleic acid amplification to form at least one separate, detectable colony of said nucleic acid product in at least one of said first and second matrix layers.

22. The method according to claim 21 wherein said first and second matrix layers each have a thickness of 50 μ

m to 10 mm.

23. The method according to claim 21 wherein said second portion of the reaction components of said amplification system includes all enzymes of said amplification system.

24. The method according to claim 23 wherein said amplification system is a Viral RNA Polymerase system.

25. The method according to claim 23 wherein said amplification is a 3SR Amplification system.

26. The method according to claim 23 wherein during said step (d) of incubating, said second matrix layer is contacted with at least one blotting membrane, and amplified product transfers to said at least one blotting membrane.

27. A method of exponential nucleic acid amplification to form detectable colonies of nucleic acids comprising the steps of

(a) providing a first solid, water-insoluble matrix layer having an average pore size ranging from 100 μ

m to 5 nm and containing a first portion of the components of a cell-free enzymatic, exponential nucleic acid amplification system;

(b) providing a second solid, water-insoluble matrix layer having an average pore size ranging from 100 μ

m to 5 nm and containing a second portion of the components of said amplification system, said first and second portions together comprising said amplification system;

(c) distributing on at least one of said matrix layers nucleic acid molecules, at least one of which may comprise a template for said amplification system;

(d) contacting said first and second matrix layers, sandwiching said nucleic acid molecules between said layers;

(e) incubating said first and second matrix layers while maintaining contact therebetween under conditions promoting synthesis of an exponentially amplified nucleic acid product by said amplification system from said at least one template,

wherein said first and second matrix layers are stable under said conditions;

wherein said conditions cause at least said first portion or said second portion of said amplification to diffuse from one of said matrix layers into the other, together with said nucleic acid molecules; and

wherein said distribution of nucleic acid molecules separates individual templates, resulting in nucleic acid amplification to form at least one separate, detectable colony of said nucleic acid product in at least one of said first and second matrix layers.

28. The method according to claim 27 wherein the first and second matrix layers each have a thickness of 50 μ

m to 100 mm.

29. The method according to claim 27 wherein said amplification system is a Viral RNA Polymerase system.

30. The method according to claim 27 wherein said amplification is a 3SR Amplification system.

31. The method according to claim 27 wherein said second portion of the reaction components of said amplification system includes all enzymes of said amplification system and wherein during said step (e) of incubating, said second matrix layer is contacted with at least on blotting membrane, and amplified product transfers to said at least one blotting membrane.

32. A method of exponential nucleic acid amplification to form detectable colonies of nucleic acids using an isothermal, cell-free, enzymatic, exponential nucleic acid amplification process, comprising the steps of

(a) mixing with a gel-producing solution a nucleic acid amplification system for said amplification process, said amplification system including at least one caged nucleotide;

(b) casting from said gel-producing solution a first solid, water-insoluble gel matrix layer having an average pore size ranging from 5 nm to 100 μ

m, said matrix completely entrapping said amplification system;

(c) distributing in said amplification system nucleic acid molecules, at least one of which may comprise a template for said amplification system;

(d) releasing said at least one caged nucleotide; and

(e) incubating said matrix layer containing said distribution molecules under conditions promoting synthesis of an exponentially amplified product by said amplification system,

wherein said matrix is stable under said conditions and wherein said distribution of nucleic acid molecules separates individual templates, resulting in nucleic acid amplification of the templates to form at least one separate, detectable colony of said nucleic acid product in said first matrix layer.

33. The method according to claim 32 wherein said first matrix layer has a thickness of from 50 μ

m to 10 mm.

34. The method according to claim 32 wherein said amplification system is a Viral RNA Polymerase system.

35. The method according to claim 32 wherein said amplification system is a 3SR Amplification system.

36. The method according to claim 32 wherein during said step (e) of incubating, said first matrix layer is contacted with at least one blotting membrane, and amplified product transfers to said at least one blotting membrane.

37. The method according to claim 32 wherein said step (c) of distributing nucleic acid molecules in the amplification system precedes said step (b) of casting the matrix from the solution containing the amplification system.

38. A method of exponential nucleic acid amplification comprising the steps of

(a) supplying a cartridge containing hollow fibers permeable to nucleotide substrates but impermeable to enzymes, said cartridge having a first volume inside said hollow fibers and a second volume surrounding said fibers;

(b) mixing with a gel-producing solution

(i) a first portion of the components of a cell-free, enzymatic, exponential nucleic acid amplification system, said first portion including all enzymes of said amplification system, and

(ii) nucleic acid molecules, at least of which may comprise a template for said amplification system;

(c) casting the gel-producing solution resulting from step (b) in one of said first and second volumes;

(d) adding to the other of said first and second volumes a second portion of the components of said amplification system, said second portion including nucleotide substrates of said amplification system, said first and second portions together comprising said amplification system;

(e) incubating said cartridge under conditions promoting synthesis of an exponentially amplified nucleic acid product by said amplification system from said at least one template in said cast mixture;

(f) melting said cast mixture;

(g) removing the melted mixture from the cartridge; and

(h) recovering amplified nucleic acid product from said mixture,

wherein said gel cast in step (c) is stable under said incubating conditions.