Method for amplification and expression of nucleic acids in solid media and its application for nucleic acid cloning and diagnostics

• US 5,958,698 A
• Filed: 08/17/1998
• Issued: 09/28/1999
• 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:
  US09/135,446
• Time in Patent Office: 407 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/299
• 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 method of detecting a nucleic acid sequence in a sample that may contain said sequence comprising the steps of:

(a) providing a cell-free, enzymatic, exponential amplification system;

(b) forming a liquid mixture of the sample and said amplification system;

(c) entrapping said liquid within solid surfaces comprising a thin layer;

(d) incubating said trapped mixture under conditions promoting synthesis of an exponentially amplified nucleic acid product from said nucleic acid sequence; and

(e) screening to detect said amplified product,

wherein the average distance between the nearest solid surfaces is smaller than the distance which the synthesized nucleic acid product can migrate by diffusion during the reaction, and

wherein copies of said nucleic acid sequence, if present in said sample, are sufficiently widely distributed in said liquid mixture to produce separate, detectable colonies of the synthesized nucleic acid product.

2. The method of claim 1, wherein said average distance is from 5 nm to 100 μ

m.

3. The method of claim 2, wherein said solid surfaces comprise a solid matrix selected from the group consisting of agarose, polyacrylamide, nylon, gelatin, alginate, carrageenan, cellulose, silica gel, titanium sponge, dextran, and polyethylene glycol.

4. The method of claim 3, wherein said matrix is chemically modified with hydrophobic and positively charged groups.

5. The method of claim 3, wherein said matrix is chemically modified with weak cationic groups selected from the group consisting of secondary and tertiary amino groups.

6. The method of claim 3, wherein said matrix has a thickness of from 1 μ

m to 10 mm.

7. The method of claim 1, wherein said step of screening comprises visualizing detectable colonies with an intercalating dye.

8. The method claim 1, wherein said step of screening comprises the steps of:

(a) expressing the synthesized nucleic acid product; and

(b) identifying an expression product.

9. The method of claim 8, wherein said expressing is by a cell-free expression system.

10. The method of claim 8, wherein the step of identifying an expression product is performed in the immobilized medium by carrying out a reaction selected from the group consisting of a specific enzymatic reaction, binding of a specific ligand, and a specific immunochemical reaction.

11. The method of claim 1, wherein said amplification system includes at least one nucleoside triphosphate bearing a label and said step of screening comprises detecting said label.

12. The method of claim 1, wherein said step of screening comprises contacting said synthesized nucleic acid product with a labeled oligonucleotide probe and detecting said label.

13. The method of claim 1, wherein said amplification system is a Viral RNA Polymerase system.

14. The method of claim 1, wherein said amplification system is a 3SR Amplification system.

15. The method of claim 1, wherein said amplification system is a Polymerase Chain Reaction system and wherein said step (c) comprises cycling the medium through at least 2 temperatures.

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

17. A method of detecting a nucleic acid sequence in a sample that may contain said sequence 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

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

m to 5 nm, completely entrapping said liquid phase,

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

(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; and

(d) screening said colonies,

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.

18. The method of claim 17, wherein the immobilized medium is shaped into at least one thin layer.

19. The method of claim 17, wherein said amplification system is a Viral Polymerase system.

20. The method of claim 17, wherein said amplification is a Polymerase Chain Reaction system and wherein step (c) includes cycling the medium through at least two temperatures.

21. The method of claim 17, wherein step (a) precedes step (b).

22. The method of claim 17, wherein said screening is in a screening medium, said screening occurring after transfer of said at least one colony from said immobilized medium to said expression medium.

23. The method of claim 17, wherein said step of screening comprises hybridizing with a labeled oligonucleotide probe and detecting said label.

24. A method of detecting a nucleic acid sequence in a sample that may contain said sequence 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 last one template; and

(f) screening said nucleic acid product,

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.

25. The method of claim 24, 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 one blotting membrane, and amplified product transfers to said at least one blotting membrane.