Method for amplification of nucleic acids in solid media

We claim:

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 .mu.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 .mu.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 .mu.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 .mu.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 .mu.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 .mu.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 .mu.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 .mu.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 .mu.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 .mu.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 .mu.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 .mu.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.

Description Submit all comments and votes

FIELD OF THE INVENTION

This invention is in the field of amplification, expression, cloning, and diagnostics of nucleic acids.

BACKGROUND OF THE INVENTION

Several methods for exponential amplification of nucleic acids in vitro have been invented so far. These are: RNA amplification by viral RNA-directed RNA polymerases (VRP), DNA amplification in polymerase chain reaction (PCR), and isothermal multienzyme (3SR) amplification of nucleic acids. In contradistinction to linear amplification, such as which takes place during RNA synthesis with a DNA-directed RNA polymerase, the number of nucleic acid molecules increases in an exponential amplification reaction as an exponential function of the elapsed time, thus allowing a large amount of nucleic acid to be obtained in a short time period starting with a low number of nucleic acid templates. Currently, an exponential amplification reaction is carried out in a liquid reaction medium that contains the components of a cell-free enzyme system comprising a reaction buffer, appropriate enzyme(s), nucleotide substrates, and, when required, polymerization primers. In this format, the product nucleic acids synthesized on each template are allowed to spread all over the reaction medium.

In VRP reaction, exponential synthesis occurs because the product and template RNAs remain single-stranded during RNA synthesis, and both serve as equally effective templates in the next round of synthesis. Thus, the number of templates doubles after each round of replication unless RNA is in molar excess over the replicase [Haruna, I. and Spiegelman, S. (1965). Autocatalytic Synthesis of a Viral RNA in vitro. Science 150, 884-886; Kamen, R. I. (1975). Structure and Function of the Q.beta. RNA Replicase. In RNA Phages (Zinder, N. D., ed.), pp. 203-234, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]. Viral RNA-directed RNA polymerases, such as Q.beta. replicase, demonstrate a strict template specificity which is achieved through recognition by the enzyme of special structures that are present in their specific templates and are absent in other RNAs. RNAs containing these structures are called "replicating RNAs". Munishkin, A. V., Voronin, L. A., Ugarov, V. I., Bondareva, L. A., Chetverina, H. V. and Chetverin, A. B. (1991). Efficient Templates for Q.beta. replicase are Formed by Recombination from Heterologous Sequences. J. Mol. Biol. 221, 463-472. Foreign nucleic acid can be made amplifiable with a VRP by providing the nucleic acid in the form of replicating RNA, for example, by inserting the corresponding nucleotide sequence into a naturally occurring replicating RNA [Miele, A. A., Mills, D. R. and Kramer, F. R. (1983). Autocatalytic Replication of a Recombinant RNA. J. Mol. Biol. 171, 281-295; Wu, Y., Zhang, D. and Kramer, F. R. (1992). Amplifiable Messenger RNA. Proc. Natl. Acad. Sci. U.S.A., in press]. VRP reaction is carried out at a constant temperature and is very fast: in a 30-min incubation, the number of RNA templates in the Q.beta. replicase reaction increases 10.sup.9 -fold [Lizardi, P. M., Guerra, C. E., Lomeli, H., Tussie-Luna, I. and Kramer, F. R. (1988). Exponential Amplification of Recombinant-RNA Hybridization Probes. Bio/Technology 6, 1197-1202].

PCR is used for the in vitro amplification of DNA. This reaction requires the annealing and enzymatic extension of two oligonucleotide primers that embrace a region within a DNA molecule to be amplified (a target region), and that use complementary strands of the DNA as templates for extension by a DNA polymerase, their growing 3' termini being directed towards each other. The word "embrace" is used here to describe the property of the primers to anneal on complementary strands of the DNA downstream from the target region. Unlike VRP reaction, the product strand in PCR is involved in a duplex with the template. Therefore, the template and product strands have to be melted apart at elevated temperature to initiate the next round of replication where each strand anneals with one of the primers and serves as a template for additional replication. The process is repeated many times by cycling between the annealing and melting temperatures, resulting in an exponential amplification of the target region [Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A. and Arnheim, N. (1985). Enzymatic Amplification of .beta.-Globin Genomic Sequence and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia. Science 230, 1350-1354; Mullis, K. B. and Faloona, F. A. (1987). Specific Synthesis of DNA in vitro via a Polymerase-catalyzed Chain Reaction. Methods Enzymol. 155, 335-350]. Currently, PCR is carried out with the use of a thermostable DNA polymerase that remains active after the reaction mixture is heated to 95.degree.-100.degree. C. to melt DNA strands apart [Erlich, H. A., Gelfand, D. and Sninsky, J. J. (1991). Recent Advances in the Polymerase Chain Reaction. Science 252, 1643-1651]. Because of the need for temperature cycling, PCR requires special equipment and is about an order of magnitude slower than VRP reaction. At the same time, PCR allows any desirable DNA to be amplified by virtue of having two specific primers, and without the need for preparing a recombinant molecule.

Recently, isothermal amplification of nucleic acids in the multienzyme system (3SR) has been invented that combines the advantages of both the VRP reaction and PCR. 3SR is based on the concerted action of three enzymes: a DNA-directed RNA polymerase, a reverse transcriptase, and RNase H, and mimics the replication system of retroviruses. A double-stranded DNA fragment containing an RNA polymerase promoter sequence at each end or a single-stranded RNA can serve as a primary template. A DNA-directed RNA polymerase, such as T7 RNA polymerase, uses double-stranded DNA molecule to linearly synthesize multiple single-stranded RNA copies of the DNA target region included between the RNA polymerase promoters. A reverse transcriptase, such as that of the avian myeloblastosis virus (AMV), makes double-stranded cDNA copies of the RNA transcripts using primers that are complementary to the 3' termini of the transcripts, and that include the RNA polymerase promoter sequence to restore the sequence at each end of the cDNAs. RNase H destroys the RNA template involved in the RNA:DNA heteroduplex after the first-strand cDNA synthesis, enabling the second strand of the cDNA to be synthesized. The action of RNA polymerase and RNase H results in the formation of single-stranded templates, allowing the amplification to proceed exponentially without temperature cycling. 3SR reaction is as fast as VRP reaction, and like PCR it is not restricted to specific templates. The product of 3SR reaction is a mixture of double-stranded DNA and single-stranded RNA molecules. Guatelli, J. C., Whitfield, K. M., Kwoh, D. Y., Barringer, K. J., Richman, D. D. and Gingeras, T. R. (1990). Isothermal, in vitro Amplification of Nucleic Acids by a Multienzyme Reaction Modeled after Retroviral Replication. Proc. Natl. Acad. Sci. U.S.A. 87, 1874-1878.

Due to the exponential nature of the amplification reactions discussed above, each of them can theoretically be employed to obtain in a short time a great number of progeny molecules starting with a single nucleic acid template. If realized, this would allow nucleic acids to be cloned in vitro, providing a powerful alternative to the conventional DNA cloning in microbial cells. Also, this would provide for an absolute method for nucleic acid diagnostics. For example, even if a single molecule of a viral, microbial, or oncogenic nucleic acid occurred in an analyzed sample, it itself, or an amplifiable reporter targeted against it [Kramer, F. R. and Lizardi, P. M. (1989). Replicatable RNA Reporters. Nature 339, 401-402], could be amplified to the amount that is easily detected by conventional techniques. However, neither of these possibilities has been so far realized because of severe practical problems.

Levisohn and Spiegelman claimed that they succeeded in the cloning of RNA molecules using Q.beta. replicase amplification system. They diluted the RNA template so that less than one molecule was expected per sample, and observed RNA synthesis in roughly half of the samples [Levisohn, R. and Spiegelman, S. (1968). The Cloning of a Self-replicating RNA Molecule. Proc. Natl. Acad. Sci. U.S.A. 60, 866-872]. However, a proper identification of the products was not performed, and the results were later diminished by the finding that RNA synthesis could be observed in Q.beta. replicase reaction even if no template were added [Sumper, M. and Luce, R. (1975). Evidence for de novo Production of Self-replicating and Environmentally Adapted RNA Structures by Bacteriophage Q.beta. Replicase. Proc. Natl. Acad. Sci. U.S.A. 72, 162-166]. Recently this spontaneous RNA synthesis was shown to be caused by replicating RNAs that contaminate the environment [Chetverin, A. B., Chetverina, H. V. and Munishkin, A. V. (1991). On the Nature of Spontaneous RNA Synthesis by Q.beta. Replicase. J. Mol. Biol. 222, 3-9]. The background from contaminating RNAs prevented the VRP-based diagnostics from being able to detect solitary nucleic acid molecules, since as many as 100 irrelevant replicating RNAs usually occurred in an average sample [Lizardi et al. (1988), Chetverin et al. (1991), supra]. The practical detection limit in VRP assays is currently 10.sup.3 to 10.sup.4 target molecules [Lomeli, H., Tyagi, S., Pritchard, C. G., Lizardi, P. M. and Kramer, F. R. (1989). Quantitative Assays Based on the Use of Replicatable Hybridization Probes. Clin. Chem. 35, 1826-1831].

Contamination problems are also encountered in PCR and 3SR reactions, although they are not so severe as for VRP reactions, since nucleic acid amplification is controlled by the specificity of the two oligonucleotide primers targeted to the template. Most significant in this case is the limited primer specificity: because of mismatches and primer heterogeneity, a non-specific priming occurs to some extent on irrelevant templates contained in the sample, and becomes competing with the specific priming as the number of specific templates goes below a certain level. At least 100 copies of a specific template are currently needed to reliably initiate PCR [Myers, T. W. and Gelfand, D. H. (1991). Reverse Transcription and DNA Amplification by a Thermus thermophilus DNA Polymerase. Biochemistry 30, 7661-7666]. It follows that neither the true cloning (i.e., obtaining the progeny of a single molecule), nor the detection of solitary molecules are currently achievable with these techniques.

There are known methods for expressing nucleic acids in vitro. During expression, the genetic information contained in a nucleotide sequence is translated into the aminoacid sequence of a polypeptide. The translation process occurs on ribosomes that use RNA as an informational messenger which is called mRNA. Translation of an mRNA can be carried out in a cell-free enzyme system comprising a reaction buffer, ribosomes, tRNAs, aminoacids, ATP, GTP, and auxiliary proteins, such as aminoacyl-tRNA synthetases and translation factors [Anderson, C. W., Straus, J. W. and Dudock, B. S. (1983). Preparation of a Cell-free Protein-synthesizing System from Wheat Germ. Methods Enzymol. 101, 635-644; Chambliss, G. H., Henkin, T. M. and Leventhal, J. M. (1983). Bacterial in vitro Protein-synthesis Systems. Methods Enzymol. 101, 598-605; Merrick, W. C. (1983). Translation of Exogenous mRNAs in Reticulocyte Lysates. Methods Enzymol. 101, 606-615]. If DNA rather than RNA is provided, it must be transcribed into RNA. In this case, the expression comprises two steps, DNA transcription and RNA translation, and can be carried out in a coupled transcription/translation cell-free enzyme system that comprises, in addition to the components of the translation system, an appropriate DNA-dependent RNA polymerase and the missing ribonucleoside triphosphates [Chen, H.-Z. and Zubay, G. (1983). Prokaryotic Coupled Transcription-translation. Methods Enzymol. 101, 674-690; Bujard, H., Gentz, R., Lanzer, M., Stueber, D., Mueller, M., Ibrahimi, I., Haeuptle, M.-T. and Dobberstein, B. (1987). A T5 Promoter-based Transcription-translation System for the Analysis of Proteins in vitro and in vivo. Methods Enzymol. 155, 416-433; Tymms, M. J. and McInnes, B. (1988). Efficient in vitro Expression of Interferon Analogs Using SP6 Polymerase and Rabbit Reticulocyte Lysate. Gene Anal. Tech. 5, 9-15; Baranov, V. I., Morozov, I. Yu., Ortlepp, S. A. and Spirin, A. S. (1989). Gene Expression in a Cell-free System on the Preparative Scale, Gene 84, 463-466; Lesley, S. A., Brow, M. A. and Burgess, R. R. (1991). Use of in vitro Protein Synthesis from Polymerase Chain Reaction-generated Templates to Study Interaction of Escherichia coli Transcription Factors with Core RNA Polymerase and for Epitope Mapping of Monoclonal Antibodies. J. Biol. Chem. 266, 2632-2638]. The known methods for expression of nucleic acids in vitro employ liquid reaction media, so that the expression products (proteins, polypeptides) can freely migrate throughout the media.

SUMMARY OF THE INVENTION

The present invention is based on our discovery that nucleic acid molecules can be, like microorganisms, grown as colonies in an immobilized medium.

According to our invention, the immobilized medium for amplification of nucleic acids comprises a liquid phase entrapped within a solid matrix that possesses a highly expanded surface with the average pore size ranging from 100 .mu.m to 5 nm, and therefore is capable of preventing convection and intermixing of different zones of the liquid phase. The immobilized medium contains an amplification system comprising a cell-free enzyme system capable of exponentially amplifying the nucleic acids. The enzyme(s) included in the amplification system can be either present in the liquid phase or immobilized on the solid matrix.

Our invention includes a method for amplification of nucleic acids in an immobilized medium. According to the invention, a nucleic acid or a mixture of nucleic acids to be amplified is introduced into the medium during, or subsequent to, its immobilization. The nucleic acid molecules become entrapped somewhere in the medium, and their exponential amplification results in a "colony" within a limited zone surrounding the progenitor template. Each nucleic acid colony comprises individual clone, i.e. the progeny of a single molecule. The method can employ any system of exponential amplification of nucleic acids in vitro, such as VRP reaction, PCR, or 3SR reaction. Provided that the nucleic acid sample has been properly diluted, different colonies occupy separate zones within the immobilized medium, and this allows the respective clones to be observed and handled separately. The method allows nucleic acids to be cloned in vitro, and even solitary molecules of specific nucleic acids to be detected in an analyzed sample, despite contamination of the sample or the medium with irrelevant templates or the occurrence of non-specific priming.

According to the preferred embodiment, amplification of nucleic acids is carried out in at least one thin layer of an immobilized medium, so that the growing colonies are arranged in a two-dimensional pattern. Employing thin layers makes it easier to separately observe and handle individual colonies, and allows replicas of the colony pattern to be prepared, for example, by colony transfer onto a blotting membrane. The replicas can be used for screening the colonies or for any other purpose, or can be stored for further use. If an immobilized medium comprises more than one layer (a sandwich medium), the enzymes and substrates included in the amplification system can be introduced into separate layers, so that the amplification reaction can be initiated at a pre-selected time by contacting the layers. By using a sandwich medium it is also possible to prepare one or more replicas while amplifying nucleic acids and to supplement the medium with new components at a pre-determined reaction time.

According to the preferred embodiment of our method, the solid matrix that immobilizes the medium should be capable of reversible interaction with nucleic acids. The use of matrices modified with positively charged and/or moderately hydrophobic groups is therefore preferred. Such matrices retard the spreading of the colonies caused by diffusion and thus increase the resolving power of the method, especially when amplifying small nucleic acids.

Entrapment of nucleic acids in an immobilized medium according to the invention substantially prevents a competition between different templates, since their progeny is not allowed to spread all over the reaction volume. In particular, this allows individual nucleic acids to be amplified in bulk with the interference from background growth being largely suppressed.

Our invention includes applications of the method for amplification of nucleic acids in immobilized media for extremely sensitive diagnostics of nucleic acids, such as nucleic acids related to viruses, microorganisms, and oncogenes. Nucleic acids in a sample, their segments, or amplifiable reporters generated in the sample in a target-dependent reaction, are amplified in a thin layer of an immobilized medium. The colonies comprised of the nucleic acids containing a particular sequence are then identified, e.g. by hybridizing them with a specific labeled probe. Solitary target molecules can be reliably detected by employing this method, even if the background from unrelated nucleic acids is several orders of magnitude greater.

Our invention includes applications of the method for amplification of nucleic acids in immobilized media for cloning nucleic acids in vitro. According to the invention, nucleic acids are amplified in a thin layer of an immobilized medium to produce colonies. The colonies are screened, for example, by transferring them onto a blotting membrane and hybridizing the membrane with a labeled probe. Alternatively, the colonies are screened by virtue of expressing nucleic acids in a protein synthesis system in vitro.

Our invention also includes methods for expression of nucleic acids in an immobilized medium. The immobilized medium for expressing nucleic acids according to the invention is similar to the immobilized medium used for nucleic acid amplification, with the exception that it contains-an expression system comprising a cell-free enzyme system capable of expressing the nucleic acids. The nucleic acids amplified according to this invention or those obtained by any other method can be expressed in the immobilized medium. According to our invention, nucleic acids can also be both amplified and expressed in the same immobilized medium. In this case, the medium contains both the components of an amplification system and of an appropriate expression system. According to the preferred embodiment, nucleic acids are expressed (or both amplified and expressed) in at least one thin layer of an immobilized medium that contains all the components of the protein synthesis system. Expressing nucleic acids in an immobilized medium results in the expression products being accumulated within the zones where the nucleic acid templates are entrapped. Employing immobilized media allows nucleic acid colonies to be easily screened for their expression products either in situ or upon their transfer onto an expression medium with a replica, or a number of nucleic acid samples to be tested in parallel in a single expression reaction. The synthesized polypeptides can be identified in situ or on a replica by an immunoassay, or by their ability to perform specific enzymatic reactions or to bind a specific ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows, schematically, growing nucleic acid colonies in an immobilized medium.

FIG. 2 shows RNA colonies grown in thin layers containing the Q.beta. replicase enzyme system.

FIG. 3 shows, schematically, a target-dependent formation of replicatable RNA reporters.

DETAILED DESCRIPTION OF THE INVENTION

The techniques and materials described herein with respect to one embodiment may not be explicitly described in other embodiments. Their application to the several embodiments described herein, however, is understood. All periodicals, patents and other references cited herein are hereby incorporated by reference.

According to our invention, exponential amplification of nucleic acids and/or their expression (hereinafter referred to as a "reaction") is carried out in an immobilized medium, rather than in solution as it is done in the prior art, in order to keep the templates and the reaction products within limited zones at fixed locations of the reaction volume.

An immobilized medium suitable for the reaction according to the invention comprises a liquid water-based phase entrapped within a solid matrix. The solid matrix has a highly expanded surface that penetrates the liquid phase, so that the liquid phase gets substantially motionless (lack of convection and intermixing of different zones of the liquid phase), apparently due to the intermolecular friction and water structuring near the surface. The solid matrix can possess a various texture, such as porous, fibrous, reticulated, coiled, capillary, lamellar, or folded, with the average distance between the nearest surfaces (the "pore" size) ranging from about 100 .mu.m to 5 nm. The upper limit of the pore size should be less then the distance at which the synthesized nucleic acids or proteins can migrate by diffusion during the reaction. (For example, nucleic acids that are about 100 nucleotides in length diffuse at the rate of approximately 1 mm per hour at room temperature, judging from the size of the colonies seen in FIGS. 2A to 2C.) The lower limit should slightly exceed the size of the nucleic acid and/or protein molecules involved in the reaction to ensure their motion required for the reaction. The matrix can be comprised of a three-dimensional network formed by polymeric molecules or their aggregates; pelleted, cemented, sintered, or compressed water-insoluble powder, grains or microcrystals; spongy substances; tightly packed fibers, microcapillaries or folded pellicle, such as a thin polymeric film or metal foil. The materials used for preparing the solid matrix should be chemically inert under the reaction conditions, and the surface should not cause denaturing of the enzymes, as by their irreversible sorption and unfolding. Various organic or inorganic substances used in biotechnology as solid supports for chromatography or electrophoresis of biopolymers, for enzyme or cell immobilization, as well as for growing bacteria, cells and viruses; such as agarose, polyacrylamide, nylon, gelatin, alginate, carrageenan, cellulose, silica gel, titanium sponge, cross-linked agarose, dextran or polyethylene glycol, and their combinations and derivatives are suitable [Primrose, S. B. (1987). Modern Biotechnology. Blackwell Scientific Publications, Oxford; Osterman, L. A. (1986). Methods of Protein and Nucleic Acid Research, Vol. 3. Springer-Verlag, N.Y.; Scopes, R. K. (1982). Protein Purification: Principles and Practice. Springer-Verlag N.Y.; Golubev, V. N., Meteshkin, Yu. V., Kananykhina, E. N., Antonyuk, V. P. and Koshel, M. I. (1989). U.S.S.R. Patent SU 1472505 A1]. When using a new type of the solid matrix, preliminary experiments should be carried out to check whether nucleic acids and/or proteins can be synthesized in the immobilized medium to a detectable level. The choice of a solid matrix also depends on a particular application. For example, temperature-resistant media should be used to carry out PCR. In this case, matrices such as comprised of polyacrylamide, cellulose, polyamide (nylon), or of cross-linked agarose, dextran or polyethylene glycol, are appropriate.

The immobilized medium according to our invention also contains the components of an amplification and/or an expression system comprising the components of a cell-free enzyme system capable of exponential amplification and/or of expression of nucleic acids, respectively. Such cell-free enzyme systems are well known in the art, and are discussed above. Enzymes included in the enzyme systems can be either dissolved in the liquid phase, or be immobilized on the solid matrix [Klibanov, A. M. (1983). Immobilized Enzymes and Cells as Practical Catalysts. Science 219, 722-727].

During amplification in an immobilized medium, nucleic acids grow as colonies rather than spread throughout the reaction volume, as schematically illustrated in FIG. 1. The upper diagram Shows segment 11 of an immobilized medium comprising solid matrix 12, and liquid phase 13 wherein the matrix is immersed and which contains reaction buffer and nucleotide substrates (not shown), enzyme molecules 14 and nucleic acid templates 15. After incubating the immobilized medium for a period of time at a temperature regime allowing nucleic acids to be amplified, nucleic acid colonies 16 form at the locations where the parental nucleic acid templates have been entrapped (the lower diagram). Similarly, during expression of nucleic acids in an immobilized medium, the expression products are accumulated at the locations where the expressed templates have been entrapped.

When carrying out amplification, it is preferred that the solid matrix is capable of reversible interactions with the nucleic acid molecules. Such interactions restrain diffusion of nucleic acids in the liquid phase, resulting in colonies with a smaller size, sharper edges, and higher concentration of nucleic acids. Most hydrophillic matrices are capable of interacting with nucleic acids to some, though different, extent due to hydrogen bonding. This ability can be further increased by chemically modifying the solid matrix with positively charged groups (providing for ionic interactions with phosphate residues in nucleic acids), and/or moderately hydrophobic groups (providing for interactions with purine and pyrimidine rings; care should be taken that the presence of hydrophobic groups does not cause denaturing of the enzymes). At the same time, the interactions should not be too strong, and the modification should not be too extensive, so that the immobilization of the nucleic acids on the solid matrix is readily reversible; otherwise, nucleic acid strands may become unavailable as templates for the amplification reaction. For example, modification of the solid matrix with weak cationic groups, such as secondary or tertiary amino groups (e.g. ethylaminoethyl, or diethylaminoethyl), is appropriate. The solid matrix can also be modified with intercalating dyes such as ethidium or propydium groups [Baidus, A. N., Babaeva, P. V., Remnev, Yu. V., Kolombet, L. V. and Borovik, R. V. (1989). U.S.S.R. Patent SU 1230187 A1]. These groups are capable of hydrophobic interactions with the stacked bases of nucleic acids and are positively charged. Moreover, the presence of these groups enables the growing nucleic acid colonies to be detected by fluorescence (see below). Methods of modifying solid matrices with various chemical groups are known in the art, and a variety of modified polymers is commercially available. Many suitable solid matrices and their derivatives compatible with enzymes and nucleic acids are widely used in liquid chromatography of biopolymers [Osterman, L. A. (1986), Scopes, R. K. (1982), supra].

Any mobile liquid outside the immobilized medium should preferably be eliminated, so that nucleic acid or protein molecules cannot be transferred to another location during the reaction. For example, water should not be allowed to condense on the surface of the immobilized medium. At the same time, the immobilized medium should not be allowed to get too dry, as this can result in inactivation of the enzyme(s). Drying up of the immobilized medium during the reaction can be prevented by carrying out the reaction in a properly closed chamber or in a cassette, by wrapping or sealing the medium with a film, or by overlaying the medium with an oil.

It is preferred to suppress the reaction until the medium gets immobilized, especially in the case of an amplification reaction; otherwise the reaction products will be prematurely synthesized and spread throughout the medium. This requirement can easily be met in the case of PCR, as this reaction does not proceed until temperature cycling is started. When carrying out an isothermal reaction, such as 3SR or VRP reaction, or an expression reaction, this can be achieved by either preparing the medium at or close to 0.degree. C. when the reaction is very slow; or keeping the reaction substrates and/or nucleic acids away from the enzyme(s) until the medium is immobilized; or having the reaction substrate(s) in a chemically unavailable "caged" form, which can be decomposed to release the normal substrate(s). An example of caged substrate is a photosensitive derivative of ATP, wherein the .gamma.-phosphate is modified with a 1-(2-nitro)phenylethyl group [Kaplan, J. H., Forbush, B., III, and Hoffman, J. F. (1978). Rapid Photolytic Release of Adenosine 5'-Triphosphate from a Protected Analogue: Utilization by the Na:K Pump of Human Red Blood Cell Ghosts. Biochemistry 17, 1929-1935] that can be converted into ATP by photolysis to initiate the reaction.

Depending on the application, the medium can be shaped into a thick bed, or into a thin layer.

Carrying out a reaction in a thick bed (packed in any suitable container such as a tube, a flask, a column, or a cartridge) can be useful when the purpose of the procedure is mere propagation, of, e.g., cloned nucleic acids. The purpose of employing an immobilized medium in this case is to minimize the interference from the background growth, especially if contaminating nucleic acids are more efficient templates than those to be propagated. This is especially important if an amplification reaction is primer-independent, such as the VRP reaction. Carrying out the reaction in an immobilized medium allows the contaminating nucleic acids to be amplified at, and their competition to the growth of the desired nucleic acids be restricted to, limited zones of an immobilized medium where the contaminating templates were originally entrapped.

It is preferred that the thick bed be prepared in such a manner that enzymes (in the examples below understood as together with nucleic acid templates and all other macromolecular reaction components, which is understood in the examples below) and substrates are present in separate zones that are shaped into granules, lamellae, or filaments and surround each other. In this format, the enzymes and substrates are kept apart during medium preparation, and the reaction starts simultaneously throughout the bed volume as the substrates begin to penetrate into the enzyme zones. Examples of such embodiments are the following.

(a) A first solution containing melted agarose and a buffer is mixed at .apprxeq.40.degree. C. with enzymes and, while liquid, is dropped into a cold buffer to form beads. Alternatively, enzyme-containing agarose is cast in a bed which is then crushed into small granules. Enzyme-containing beads can also be prepared by soaking a commercially available beaded agarose in an appropriate solution, or by dispersion of an agarose:enzyme solution in an oil, as described for the entrapment of living cells [Nilsson, K., Brodelius, P., and Mosbach, K. (1987). Entrapment of Microbial and Plant Cells in Beaded Polymers. Methods Enzymol. 135, 222-230]. The cold drained granules containing enzyme(s) are then poured into a second agarose solution containing reaction substrates and is pre-cooled but still liquid, and which is then placed on ice to ensure rapid gelling of agarose. (Of course, the reversed format can also be used, i.e. fusion of the granules containing nucleotides into the agarose containing enzyme(s) and template.) The immobilized medium is incubated at 20.degree. to 37.degree. C. to allow the reaction to proceed, and the reaction product are eluted or extracted by an appropriate method known in the art.

(b) The enzyme-containing granules described in (a) are coated with alginate shells by including sodium alginate into the first agarose solution and calcium ions into the buffer whereinto the solution is dropped, or by soaking the already formed granules in a solution containing alginic acid, and then pouring the drained granules into a Ca.sup.2+ -containing buffer. Alginate shells can be further hardened by treating them with polyethylene imine. Bucke, C. (1987). Cell Immobilization in Calcium Alginate. Methods Enzymol. 135, 175-189. The granules can also be coated with kappa-carrageenan [Chibata, I., Tosa, T., Sato, T. and Takata, I. (1987). Immobilization of Cells in Carrageenan. Methods Enzymol. 135, 189-198], or with cellulose nitrate, nylon, and other types of semipermeable membranes [Chang, T. M. S. (1976). Microencapsulation of Enzymes and Biologicals. Methods Enzymol. 44, 201-218]. The reaction is then carried out in the coated granules (capsules) by incubating them in a substrate-containing solution. In contrast to sma