Solid medium for amplification and expression of nucleic acids as colonies

• US 6,001,568 A
• Filed: 09/30/1996
• Issued: 12/14/1999
• Est. Priority Date: 10/26/1992
• Status: Expired due to Term

• Current Assignee: Institut Belka
• Original Assignee: Institut Belka
• Inventors: Chetverina, Helena Vladimirovna, Chetverin, Alexander Borisovich
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/723,260
• Time in Patent Office: 1,170 Days
• Field of Search: 435/6, 435/91.2, 435/69.1, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

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 preformed immobilized medium suitable for producing, from individual nucleic acid molecules applied thereto, separate detectable colonies by a cell-free enzymatic exponential amplification process, comprising

a) an aqueous liquid phase that includes a cell-free nucleic acid polymerase enzyme system capable of performing said process, and

b) a thin layer, from 1 μ

m to 10 mm in thickness, of a solid, water-insoluble matrix having an average pore size ranging from 100 μ

m to 5 nm, completely entrapping said liquid phase.

2. The medium 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.

3. The medium according to claim 1, wherein said matrix is chemically modified with hydrophobic and positively charged groups.

4. The medium according to claim 1, wherein said thin layer has a thickness in the range of 50 μ

m to 1 mm.

5. The medium according to claim 1 wherein said enzyme system includes at least one labeled nucleotide substrate.

6. The medium according to claim 1 wherein said cell-free system comprises at least one caged nucleotide substrate.

7. The medium according to claim 6 wherein said cell-free system is an isothermal amplification system selected from the group consisting of a Viral RNA Polymerase system and a 3SR Amplification system.

8. The medium according to claim 1 wherein at least one enzyme is attached to said matrix.

9. The medium according to claim 1 further including an activatable reagent for preventing nucleic acids from serving as templates for enzymatic amplification.

10. The medium according to claim 1 wherein said enzyme system is a Polymerase Chain Reaction system.

11. A preformed solid, water-insoluble matrix, for use in a cell-free enzymatic expontetial amplification of individual nucleic acid molecules having a thickness of from 1 μ

m to 10 mm and an average pore size of from 100 μ

m to 5 nm free of said nucleic acid molecules and completing entrapping an aqueous liquid phase that includes at least the enzyme components, including a nucleic acid polymerase, of a cell-free exponential nucleic-acid amplification system but fewer than all necessary components of said system.

12. The matrix according to claim 11 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.

13. The matrix according to claim 11 wherein said thickness is in the range of 50 μ

m to 1 mm.

14. The matrix according to claim 11 wherein said exponential amplification system is an isothermal amplification system.

15. The matrix according to claim 11 further including an activatable reagent for preventing nucleic acids from serving as templates for enzymatic amplification.

16. A preformed enzyme-free solid, water-insoluble matrix having a thickness of 0.1 μ

m to 10 mm, having an average pore size of from 100 μ

m to 5 nm, and completely entrapping nucleotide substrates for a cell-free enzymatic exponential amplification system.

17. The matrix according to claim 16 wherein said matrix is a nylon membrane.

18. The matrix according to claim 17 wherein said matrix is dry.

19. A multi-layer system of at least two preformed layers for performing a cell-free enzymates nucleic acid exponential amplification process, comprising

a) a first layer comprising a solid, water-insoluble matrix having a thickness of from 1 μ

m to 10 mm and an average pore size of from 100 μ

m to 5 nm, completely entrapping a first, incomplete portion of the ingredients of a nucleic acid polymerase enzyme system capable of performing said process,

b) a second layer apart from but stackable with said first layer, comprising a solid, water-insoluble matrix having a thickness of from 1 μ

m to 10 mm and an average pore size of from 100 μ

m to 5 nm, completely entrapping a second, incomplete portion of said ingredients,

wherein neither said first nor said second layer alone is capable of performing said process but, when stacked, diffusion between said layers creates an immobilized medium capable of producing, from individual nucleic acid molecules distributed on one of said layers, separate detectable colonies by means of exponential amplification.

20. The system according to claim 19 wherein the matrix of said first layer completely entraps a liquid phase that includes all enzymes for performing said process and wherein the matrix of said second layer is dry and contains at least one nucleotide substrate ingredient for performing said process.

21. The system according to claim 19 wherein said enzyme system comprises nucleotide substrates selected from the group consisting of ribonucleotides, deoxyribonucleotides and both ribonucleotides and deoxribonucleotides.

22. The system according to claim 19 wherein the matrices of said first and second layers are selected from the group consisting of agarose, polyacrylamide, nylon, gelatin, alginate, carrageenan, cellulose, silica gel, titanium sponge, dextran, and polyethylene glycol.

23. The combination according to claim 19 wherein at least one of the matrices of said first and second layers is chemically modified with hydrophobic and positively charged groups.

24. The system according to claim 19 further comprising at least one blotting membrane.

25. The system according to claim 19 further comprising a third layer including a completely entrapped second liquid phase comprising a cell-free expression system.

26. The system according to claim 19 wherein at least one of said first and second layers has a thickness in the range of 50 μ

m to 1 mm.