CA2037673A1 - Ribonuclease scission chain reaction - Google Patents

Abstract

ABSTRACT
The present invention is directed to a process for amplifying the signal generated from the hybridization of a DNA sequence in a sample. The sample is mixed with a molar excess of RNA which is complementary to the DNA sequence to be detected, under conditions such that a DNA-RNA hybrid is formed. The DNA-RNA hybrid is treated with an enzyme,i.e. ribonuclease H, which is capable of digesting the RNA of said DNA-RNA hybrid into low molecular weight unhybridized RNA fragments. The process repeats itself generating large quantities of unhybridized RNA fragments which can readily be detected. The present invention as application in diagnosis of infectious disease,genetic disorders or cellular diseases.

Description

-1- 2~37~7~

RIBONUCL~:ASE SCISSION CHAIN REACTION

BACKGROUND OF THE INVENTION

The present invention relates to methods for detecting nucleic acid sequences. In particular, the invention concerns methods for detecting the presence or absence of a particular nucleic acid sequence by amplifying the signal generate~ from the hybridization of a specific nucleic acid with a molar excess of complementary RNA.
Nucleic acid hybridization is a common technique in a molecular biology laboratory. Application of this technique has been extended to clinical laboratories for the identification of genetic defects and diagnosis of infectious diseases.
N~cleic acid hybridization methods provide assay for detecting nucleic acid sequences of medical significance, such as DNA or RNA sequences indicative of genetic diseases, cancer, and bacterial and viral infections. Nucle~c acid hybridization assays are based on the very specific base pairing that is found in hybrids of DNA and RNA. Base sequences of analytical interest appearing along a strand of nucleic acid can be detected very specifically and sensitively by observing the formation of hybrids in the presence of a probe nucleic acid known to comprise a base sequence that is complementary with the sequence of interest.
Various infectious diseases can be diagnosed by the presence in clinical samples of specific DNA
sequences characteristic of the causative microorganism. These include bacteria, such as Salmonella, Chlamydia, and Neisseria; viruses such as the hepatitis viruses; and protozoan parasit~s, such as the Plasmodium responsible for malaria. U.S. Pat.
No. 4,358,535 issued to Falkow describes the use of specific DNA hybridization probes for the diagnosis of infectious diseases. A problem inherent in the Falkow procedure is that a relatively small number of pathogenic organisms may be present in a clinical sample from an infected patient and the DNA extracted from these may constitute only a very small fraction of the total DNA in the sample. Specific amplification of signal generated from suspected sequences prior to immobilization and hybridization detection of the DNA samples could greatly improve the sensitivity and specificity of these procedures.
Routine clinical use of DNA probes for the diagnosis of infectious diseases would be simplified considerably if non-radioactivly labeled probes could be employed as described in EP 63,879 to Ward and EP

2~3~673 0131830 to Dattagupta and Crothers. In the Ward procedure biotin-containing DNA probes are detected by chromogenic enzymes linked to avidin or biotin-specific antibodies. This type of detection is convenient, but relatively insensitive. The combination of specific DNA amplification by the present method and the use of stably labeled probes could provide the convenience and sensitivity required to make the Falkow and Ward procedures useful in a routine clinical setting.
It is evident that for hybridization assays to attain their full analytical potential, methods for increasing the sensitivity of detection even further are needed. Considerable efforts have been applied to this aspect in recent years and a number of different approaches have been conceived and developed. Particularly promising are approaches based on the biochemical amplification of the target nucleic acid sequence or its complementary signal sequence. While detection systems each have their own sensitivity limits, biochemical systems have been developed which can make millions and millions of copies of the target or signal sequenaes thereby to extend the effective sensitivity limits of such detection systems by many orders of magnitude.
Two distinct approaches have been used to increase the sensitivity of detection: (1) the ~4 2~3r~7 ~?

amplification of a particular sequences of interest (target sequence) and/or (2) the amplification of the signal generated from the target sequence.
A series of nucleic acid amplification processes presently exist. Perhaps, the most well-known process is the polymerase chain reaction method, or PCR, which is described in U.S. Pat. No. 4,683,202.
PCR employs a pair of specific oligonucleotide as primers for the two complementary strands of the double stranded form of a target sequence. T~e primers are chosen such that they form specific hybrids at the opposite 3' ends of the complementary target strands. Using a thermostable DNA polymerase, the primers are extended synthetically in correspondence with the target sequences. A thermal cycling process is required in order to form specific hybrids and, after extension, to denature the hybridized, extended strands for further primer hybridization and extension. Repeating the process several times results in a geometric amplification of the amount of the target sequences in the mixture.
A variation of PCR is the ligase chain reaction (LCR) described in European Patent Publication 320,308. This method requires at least four separate oligoprobes, two of which hybridize to opposite ends of the same target strand such that when they are hybridized to the target sequence their respective 3' 2 ~ 3 7 ~ ~ 3 and 5' ends are juxtaposed for ligation. The third and fourth probes hybridize with the first and second probes to form, upon ligation, fused probes which can be denatured and detected.
EP 0310 229 (Stanford) describes a method of amplification ~y introducing specific promoter sequences in a sample by a method similar to PCR.
This method appears to be very similar to the transcription amplification system (TAS) disclosed in W0 88/10315 (SISKA). Both methods use a half promoter on the strand containing primer sequence to initiate extension. As in PCR, TAS uses pairs of oligoprimer to hybridi~e with opposite ends of a desired target. After extension the complementary strand is produced using a target specific sequence containing a strand complementary to the already synthesized strand. The primers are selected such that the extension products comprise transcription promoter sites. After extension once or repeat extension as in PCR, multiple copies of target specific RNA transcripts are produced, using a promoter specific RNA polymerase and ribonuclease triphosphate (rNTPs), the extension products are themselves further amplified by transcription.
PCT Publication No. 88 10315 also provides a method of further amplification of the product RNA by an enzyme like Q-beta replicase. The method uses a -6- ~37~ 13 specific RNA probe whi~h i5 capable of specific transcription by a replicase enzyme.
EP 9300 796 describes the use of a restriction enzyme in conjunction with a DNA polymerase to generate primers which can be extended for amplification in a hybridization assay. Although the claims are directed to both RNA and DNA, it is not clear how specific RNA-DNA hybrid fragment are generated. If RNAse H is used, the probe is destroyed and the sample sequence has to repeat the process. In the method above described, a restriction enzyme is used to cut the sample, the digest is hybridized with a probe DNA, partially digested product is formed, the 3'hydroxyl residue of the digested DNA is then extended with a DNA
polymerase when it is hybridized with the probe. The extended product is then digested and the whole process is repeated until all the probe seguences are used up. The method is li~ear and more complicated than other known amplification methods. Although not described, in its best mode this appears to be similar to PCR where primers are generated by restriction enzyme digestion.
While all of these methods yield amplification of a target nucleic acid sequence, none are without complexities which are undesirable for the general and unsophisticated user. Many of the prior art ~33~ ~3 methods require multiple incompatible steps that can be accomplished only by cumbersome manual procedures or complex and expensive instruments for automating the many manipulations required. Further, many require the preparation of multiple sophisticated reagents which limits the ready application of the methods to different target sequences.
Peter Duck, et al, Fourth San Diego Conference, October, 1989, describe a nucleic acid probe diagnostic amplification system based on a sy~thetic DNA-RNA-DNA hybrid probe molecules. When such a unique probe is hybridized to a DN~ target, it is stable at a defined operating temperature but upon cleavage of the paired RNA portion of the probe with RNAse H, the probe fragments are destabilized as a result of their decreased melting temperature.
Consequently, the target molecules catalyze successive cleavage of probe molecules at the RNa link causing the accumulation of detectable DNA probe fragments.
A. Running, et al, Fourth San Diego Conference, October, 1989, describe chemical approaches to amplify the signal output in a nonradioisotopic DNA
probe assay, using large branched single-stranded oligodeoxyribonucleotide polymers. These branched "amplification multimers" contain a primary sequence that binds directly or indirectly to the target 2 ~ 3 nucleic acid and a set of secondary sequences complementary to a labeled oligonucleotide. First the amplification multimer is hybridized to the target then multiple copies of a short labeled probe are bound to the multimer. The signal amplification achieved is dependent upon the number of secondary sequences in the controlled network of nucleic acids.
Sharon J. Kinard, et al, Fourth San Diego Conference, October, 1989, describe a DNA probe system consisting of a family of target-dependent "primary" probes and target independent, enzyme-labeled "secondary" probes.
The prior art techniques lack the combination of simplicity and sensitivity needed for most of the applications. Recent discovery of the method of sample amplification by a polymerase chain reaction has provided a unique solution to the sensitivity problem. Yet, the desired simplificity for widespread clinical application is still not available. The present method is a solution to both sensitivity and simplicity problems for its application in a clinical laboratory.
SUMMARY OF THE INVENTION
The present invention ~s a process for amplifying the signal generated from the hybridization of at least one specific nucleic acid 2~3P~3 sequence in a sample containing a nucleic acid or mixture which comprises;
(a) mixing a sample containing at least one specific nucleic acid sequence with a molar excess of RNA which is complementary to said DNA sequence, under hybridization conditions such that a DNA-RNA
hybrid is formed, (b) treating said DNA RNA hybrid of step (a) with an enzyme capable of digesting said RNA of said DNA-RNA hybrid, to generate low molecular weight unhybridized RNA fragments from said DNA sequence resulting from step (b).
The method uses the specificity of ribonuclease H degradation of RNA in a DNA-RNA hybrid. When such an enzyme digestion is carried out under conditions of excess RNA probe and at a temperature where degraded RNA-DNA hybrid is less stable than the intact molecules the digestion will be continued until all the excess RNA probe is utilized. The resultant product of degradation are oligoribonucleotides with 5' phosphate and 3'-OH and single-stranded DNA. Since the Tm of the resultant RNA-DNA hybrid is lower than the Tm of the intact hybrid, heating the reaction mixture removes the oligoribonucleotides and regenerates the single-stranded DNA.

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The temperature of such process will depend on the size of the hybrid. If the starting probe is less than 20 nucleotides long a temperature of about 37 C or higher will be sufficient for a cycling process. Otherwise thermocycling to higher temperatures may be required.
Using RNA-DNA copolymers with the RNA portion as the probe, the amplification process can be geometrically enhanced. The geometric enhancement rate in the presence of excess enzyme is dep~de~t on the rate of formation of the hybrid.
The present method is especially useful for amplifying the signal for rare species of nucleic acid present in a mixture of nucleic acids for effective detection of such species.
In addition to detecting infectious diseases and'~
pathological abnormalities in the genome of organisms, the process herein can also be used to detect DNA polymorphism which may not be associated with any pathological state.
The present invention is related to the analysis of the presence or absence of a specific nucleic acid se~uence in a sample. ~he method can also be applied for the amplification of the signal of a sample for further processing and analysis.

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Figure 1 illustrates the steps and details of the ribonuclease scission chain (RSC) reaction.
Figure 2 illustrates the steps and details of the RSC reaction in combination with an immobilized system.
Figure 3 is a schematic drawing of the generation of plasmid pECGl.
Figure 4A and B are autoradiographs showing production of single species of RNA.
Figure 5 is an autoradigraph showing amplification of the degradation product (RNA
fragments).
Figure 6 is an autoradiograph demonstrating an immobilized DNA-RNA hybrid.

The principle of the Ribonuclease Scission Chain (RSC) reaction is iliustrated in Figure 1. In the initial reaction wherein the amount of RNA or number of RNA probes is designated by "n", single stranded DNA is mixed with a RNA probe under conditions such that a DNA-RNA hybrid is formed. The probe i8 an oligo- or polyribonucleotide ~equence and the sample is a DN~ containing material. After incubation of the DNA sample with the RNA probe under hybridization conditions a RNA-DNA hybrid is formed. The hybrid is then digested with a specific enzyme which uses RNA-DNA hybrid as the specific substrate. Such an -12- 2 ~ 3 !-~ ~ Yi ~

enzyme is known and a typical example is ribonuclease IRNAse H). When such an enzyme digestion is carried out under conditions of excess RNA probe and at a temperature where degraded RNA-DNA hybrid is less stable than the intact molecules, the digestion will be continued until all the excess RNA probe is utilized, resulting in detectable RNA fragments.
The resulting product is then analyzed for the identification of the presence of the DNA sequence complimentary to the probe RNA.
A secondary amplification can be performed using a RNA-DNA probe, wherein the RNA is linearly and covalently linked to the the DNA. After the initial RNA-DNA hybrid is digested, the product will contain a DNA attached to RNA. This product then can further react with another RNA probe system to produce more detectable product. By using sets of immobilizable or probes immobilized with sets of labeled RNA
probes, higher order amplification can be achieved.

Yet another embodiment of the present invention includes further treatment of the digested RNA-DNA
hybrid mixture with an enzyme to extend the low molecular weight RNA products~ This amplified product is then analyzed for the final se~uence identification.

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As shown in Figure 2, the RNA probe can be immobilizable or immobilized and labelled prior to or during the initial step of the RSC reaction. Once an initial DNA-RNA hybrid has formed, the presence of ribonuclease H causes the detac~ment of low molecular weight unhybridized labelled RNA fragments in solution which can be detected to determine the presenceof the analyte.
The target sequence is immobilized either before or after hybridization with the excess RNA probe and resulting immobilized hybrids are readily separated from unhybridized nucleic acids.

The Probe.

Ribonucleic acid probes can be synthesized by known methods. The probe sequence is first identified and the DNA containing that sequence is prepared by commonly known methods. The DNA sequence is then inserted in proper orientation into cloning vectors containing specific promoters. Usually vectors containing bacteriophage promoters SP6 or T7 are used. The inserted fragment containing vector is then amplified and purified. To the purified sample, specific RNA polymerase and ribonucleosidre triphosphates are added to initiate the synthesis of RNA probes. The low molecular weight probes can also 2~3767~
by synthesized chemically. Once the probe is made and purified they can be chemically modified by a number of known methods either to immobilize to a solid support or to introduce a detectable label. To synthesize mixed sequence probes one can start with the RNA probe and then add the DNA sequence either chemically or enzymatically.
Recently, a solid phase synthesis of an RNA
dodecamer has been described by Chou et al., ~IOCHEMISTRY, 28, 2422-2435 (1989). The authors present a detailed description of the synthesis of individual active mononucleotide precursors RNA for poly- or oligonucleotide synthesis in a commercially available synthesizer (Applied Biosystems' Model 380 B).
Other synthetic methods are also known in the art. Sporat and Gait describe a solid phase phosphotriester method of RNA synthesis in "Oligonucleotide Synthesis, A Practical Approach,"
M.~. Gait, Ed. IRL press, 1984, pp 83. Once the RNA
probe sequence is prepared either chemically or biochemically, they can be modified to carry a label by known methods. The labels can be directly attached to the probe or to the covalently linked DNA
region. For example, modified deoxynucleoside residues can be added synthetically or enzymatically at the 5' or 3' terminus of the RNA probe. Enzymes -15- 2~37~73 like ligase, or terminal transferases can be used for this purpose. Synthetic methods are quite well known in the art for this type of reaction. The modification can be haptens like biotin~ or a fluorescent radical like r~odamine or fluorescein, or an enzyme like horseradish peroxidase or alkaline phosphatase.
The sample.
The sample suspected or known to contain the intended target nucleic acid may be obtained from a variety of sources. It can be a biological specimen, a food or agricultural sample, an environmental sample, and so forth. In applying the present method to the detection of a particular nucleic acid sequence in the assistance of medical diagnosis, the test sample can be a body fluid or exudate such as urine, blood, milk, cerebrospinal fluid, sputum, saliva, stool, lung aspirates, throat or genital swabs, and the like. The nucleic acid or acids may be obtained from any source, for example, from plasmids such as pBR322, from cloned DNA or RNA, or from natural DNA or RNA from any source, including bacteria, yeast, viruses, and higher organisms such as plants or animals. As discussed elsewhere in more detail herein, the target nucleic acid can be RNA or DNA, including messenger RNA. In some circumstances, it will be necessary or desiràble to treat the test r~ ~ 7 3 sample to release and/or extract the target nucleic acid for hybridization, and/or, when the target nucleic acid is presented in double stranded form, to denature and render such in hybridizable single stranded form by means well known in the art. See Maniatis et al., Molecular Cloning A Laboratory Manual (New York; Cold Spring Harbor Laboratory, 1982), pp. 280-281. Although not generally necessary, it can be desirable, such as to increase the overall efficiency of hybridization, to subject the target nucleic acid to restriction digest. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized. For RNA analytes complementary DNA is produced before reaction. The specific sample nucleic acid sequence may be only a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be subjected to signal amplification be present initially in a pure form; it may be a minor fraction of a complex mixture, such as a portion of the globin gene contained in whole human DNA or a portion of nucleic acid sequence due to a particular microorganism which organism might constitute only a very minor fraction of a particular biological sample. The starting nucleic acid may contain more -17- 2~3 76 ~

than one desired specific nucleic acid sequence which may be the same or different. Therefore, the present process is useful not only for amplifying the signal generated by a specific nucleic acid sequence, but also for amplifying the signal generated from more than one different specific nucleic acid sequence located on the same or different nucleic acid molecules.
The Hvbrid.
It is well known that the thermal stability of different hybrids increases in the order DNA-DNA< DNA-RNA< RNA-RNA. This difference in property should provide better specificity and greater detection sensitivity for a mixed hybrid.
Harper, M.D., Marselle, L.M., Gallo, R.C., and Wong-Stall, F., Proc. Natl. Acad. Sci., U.S.A., 83, 772, 1986. Cox, K.H., Deleon, D.V., Angerer, L.M., and Angerer, R.C., Dev. Diol., 101, 485, 1984. RNA
as the sample and DNA as the probe have been used more often for the identification of microorganisms.
Shaw S.B., DNA probes for infectious diseases, Tenover, F.C~, Ed., CRC Press, P. 101, 1989.
DETECTION MET~OD8 Since the product of the ampli~ication is degraded RNA, determination of size by electrophoresis, for example, may be a sufficient method alternatively of detection in an immobilizable -18- ~3~ ~

probe system, release of product into solution can be monitored by absorbance, fluorescence (when probe is labelled with a therapy or other known methods.
The following examples are offered by way of illustration and are not intended in limit the invention in any manner. In these examples all percentages are by weight if for solids and by volume if for liquids, and all temperatures are in degrees Celsius unless otherwise noted.

EXAMPLE 1: RNAse H di~estion of excess RNA in presence of DNA.

The present example was carried out to demonstrate the feasibility of degradation of RNA in the presence of a small quantity of specific DNA.
Mixtures of commercially available (Sigma, St.
Louis, MO, USA) salts of POLYTHYMI~ILIC (represents DNA) and POLYADENYLIC (represents RNA) acids in different ratios were prepared in an aqueous buffer containing 20mM Hepes-KOH (pH8) 50mM KCl, lOmM MgC12 and lmM DTT. The mixtures were then heated to 950C
and cooled to 37 C. RNAse H (AMERSHAM, Arlington Heightæ, IL.)was added to the mixtures and the mixtures were incubated at 37C for 30 m~n. They were then analyzed by agarose gel electrophoresis.

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The results indicate that excess RNA can be digested in presence of small amount of DNA.
EXAMPLE 2: RNAse H degradation of_RNA in a subpicoaram amount of a DNA-RNA hybrid.
Synthesis of specific RNa by cloning and in vitro transcription:
Plasmid pSS737, described in EP 130515, was maintained and purified by P. Rae (Molecular Diognostics Inc.) and closed circular DNA samples of the plasmid were obtained from his laborator~
The plasmid contains a 737 bp fragment of a human beta globin sequence which can be released from the plasmid by Eco Rl digestion. It was gel purified, alkaline phosphatase treated and ligated into the linearized RNA promoter containing vector pSPT18. The ligation product was then transformed into DH5 cells and plated. Of the resulting 12 colonies, nine were positive in that when treated with Eco Rl, the 737 bp fragment could be released from the vector. Plasmid pSPT18 (Pharmacia) can be used to clone DNA for transcription using SP6 or T7 RNA polymerase. The 737bp Eco RI fragment was cloned into the Eco Rl cloning site of pSPT18 so that in vitro transcription products from the cloned fragment can be produced by using either SP6 or T7 RNA
polymerase. The new plasmid pECGl containing 3887 bp, which is a derivative of pSPT18 contains the 737bp 2 ~3 ~
human beta globin fragment, the SP6RNA promoter and the T7RNA promoter as shown in Figure 3. The pECGl closed circular DNA was prepared by routine plasmid preparation methods. The purified DNA was then digested with either BamH1 or Bgl I or DdeI or Bsu36I, to generate ~NA transcripts of varying lengths. For example, when pECG1 is cut with DdeI
and then transcribed using the T7RNA promoter, the resulting RNA is approximately 180 nucleotides (bases); Bsu36I would provide a resulting RN~Lof, appproximately 380 nucleotides. This is shown in Figure 4, wherein pECGl (labeled with 32P-UTP) was used as a template. Figure 4A contains pECGl cut with DdeI; Figure 4B contains pECG1 cut with Bsu36I.
Both Figure 4A and 4B show a single species RNA
transcript of a particular length. Southern blotting experiments confirmed that the RNA produced by restriction enzyme cut plasmid is specific for and complimentary to the 737 bp fragment insert. The digested DNA was purified by phenol extraction and ethanol precipitation. The DNA was then used for in vitro transcription to produce RNA specific for human beta globin sequence. A typical transcription mixture contained 80 to 100 ng of total digested DNA, .5mM concentration of each of the four n~cleoside triphosphates, UTP, ATP, GTP and CTP, units of the appropriate RNA polymerase in a buffer containing 40 mM tris HCl (pH 8), 10 ~M NaCl, and 4 mM spermidine, 10 mM dithiothreitol 10 mM MgC12. The reaction mixture also contained 32p labelled UTP or CTP. The reaction was carried out at 37C for 3 hours. After the reaction the product was purified by gel electrophoresis and electroelution. For analytical purposes formaldehyde agarose gels were used. The preparative purposed polyacrylamide gels were used.
The plasmids pSPT18 and pECGl were digested with Eco Rl and product was run on an agarose gel and transferred to nitrocellulose ~aper and hybridized with pECGl product RNA. The results showed hybridizes with the 737 fragment and the parent DNA.
The transcription mixture was then diluted to 0.3 picograms equivalent DNA specific DNA and digested with RNAse H as in example 1 and the products were analyzed by gel electrophoresis and autoradiography.
The results clearly showed that RNA in a thousand fold dilution of the transcription mixture which contains only about 0.3 picogram of the complementary DNA in the digestion solution can be degraded by the enzyme. This translates into about a million molecules in the reaction mixture. Ten to twenty-five times lower concentration can also be digested.

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EXAMPLE 3: AmPlification The oligoribonucleotides of Examples 1 and 2 can be ampified by temperature cycling the RNase H
treated RNA-DNA hybrid. By increasing the temperature, the oligoribonucleotides dissociate from the single stranded DNA and allow intact RNA to bind.
Excess RNA was prepared by invitro transcription of pECGl/DdeI DNA. The RNA was treated with DNAse I, gel purified, electroluted, and Ethanol precipitated, to insure a clean preparation. DNA was then_added back to the RNA and allowed to hybridize. After hybridization, the sample was treated with RNase H.
The temperature was then raised to 70C to dissociate the digested RNA. When the temperature is lowered to 37C hybridization of intact RNA can occur, allowing the cycle to be reinitiated. The cycling was repeated 2X, in the presence of RNase H and varying the temperature. The results are shown in Figure 5.
Lanes 1 and 2 are controls, neither contain added DNA. Lane 1 has no RNase H; Lane 2 contains RNase H.
There is no non-specific degradation of RNA by cycling or RNase H. In lanes 3 and 4, 100 ng of a 20 base oliogonucleotide (NDA 1, used in PCR) was allowed to hybridize to the RNA. In the absence of RNase H (Lane 3) there is no amplification; in the presence of RNase H (Lane 4) there is degradation of the intact RNA molecule and resultant amplification of --2 3 ~ r~ ~ rlJ 3 the degradation product. In Lanes 5 and 6j 100 ng of DNA (pECG1/DdeI DNA) was added to intact RNA and hybrized. In the absense of RNase H (Lane 5), there is no amplification; in the presence of RNase H (Lane 6), there is degradation of the intact RNA molecule and resultant amplification of the degradation product. In summary, when RNA-DNA hybrids are formed and then treated with RNase H, degradation of the specific (target) RNA results. In addition, temperature cycling increases the rate of degLradation of the target RNA.

EXAMPLE 4: Immobilized probe and secondary digestion.
Preparation of RNA-DNA conjugate:
RNA-DNA covalent conjugate was prepared by using the product RNA in the above example as primer for extension by using a polymerase. This method is known in the art. Ddel digested pECGl transcript as the primer and linearized pSS737 as the template were used. The product was then gel purified. The extension reaction can be carried out in solution or in a form where the initial primer RNA is immobilized to solid support via its 5' end. The RNA and the RNA-DNA conjugate can be immobilized either via its ends or along any RNA portion in the molecule. The digested RNA or RNA-DNA conjugate should be 2~3~7~
releasable into solution for detection. A
complementary RNA (complete or partial seuqence) to the DNA portion of DNA tailed RNA is then prepared either by cloning into pSPT18 or by synthetic method.

DNA generated by pECGl cut by DdeI as described in Example 3, was immobilized to Sephadex~particles using well know CNBr activitation methods. Excess DNA was removed by washing during preparation of Sephadex-bound DNA. RNA generated by transcrLption of the pECGl/Ddel DNA, as described in Example 3, was added to the immobilized DNA and hybrized, in the presence of IVT buffer for one hour at 80C. The RNA-DNA hybrid bound Sephadex particles were washed, twice for 30 minutes at 37C in IVT buffer and centrifuged to remove unhybridized RNA and then treated with RNase H at 37C for one hour in IVT
buffer. Both a decrease in radioactivity of the bound Sephadex particles and an increase in radioactivity of the supernate were measured.
The above experiment was conducted with 32P-pECGl/Ddel RNA was immobilized to Sephadex particles and pECGl/Ddel DNA was added and hybridized.In both experiments, there was good binding of RNA or DNA to the Sephadex particles.
Although there was release of the degradation product into the superate, strongly basic condition (i.e. I N NaOH,) enhanced the release of the degradation product ~NA. A possible suggestion is the porous nature of the Sephadex particles, which may inactivate the RNase H enzyme or harbor the digested oligoribonucleotides making release into the supernate difficult.

EXAMPLE 5: Immobilization on Nitrocellulose Standard methods (ref) were used to immobili~,ed ~00 ng pECG1/DdeI DNA onto nictrocellulose (NC). The pECG1/DdeI RNA was labelled with 32P-UTP and biotin-11-UTP during the transcription reaction. The dual label allows for detection utilizing a radioactive label and/or a biotin label, i.e.
autoradiograph and/or BRL colorimetric detection.
Figure 6 shows the results of immobilizing pECGl/DdeI
and hybridizing with 32p-biotin-pECG1/DdeI RNA. The filter 1, is in the absense of RNase H; filter 2, is in the presence of ~Nase H. From the autoradiographs, confirmed with detection of RNA, filter B demonstrates the ability to digest RNA in the presence of RNase H from an immobilized DNA-RNA
hybrid.

Preparation of the labeled probe:
(i) La~eling of RNA.

v~, ~ r There are many standard methods of labeling of RNA known in the art. For example, during the specific transcription of th template which has been cloned in the SP6 system, qv., biotin~ UTP can be used instead of UTP. This will incorporate biotin label in the probe. Other similar analogues can be used to provide other primary or secondary labels. A
5-allylamino UTP will provide a primary amine residue for subsequent labeling with a fluorophore or an enzyme. NUCLEIC ACIDS PRO9ES, R.H. SYMONS, C~ P~ESS, 1989 p. 38.Enzymes like terminal transferase can be used to incorporate labeled nucleosides to the probe.

Labeling of DNA is more well known in the art than RNA~ The probe RNA-DNA conjugate is immobilized onto one set of solid supports and the labeled DNA
complementary RNA is immobilized onto another set of solid support. The sample DNA is hybridized to the first set of support under the conditions described by Maniatis et al, Molecular cloning, Cold Spring Harbor Laboratory, 1982 P-332 using immobilized RNA
and soluble DNA.
After hybridization the support i~ washed with RNAse H digestion buffer as described in Example 1.
The washed solid material is then taken into an eppendorf tube and 12 units of RNAse H is added and digestion is carried out at 37C for 20 minutes. The ~ ~ 3 ~ ~ 7 ~
tube is centrifuged and the supernatant is added to another test tube where the solid support immobilized RNA complementary to the labeled DNA is suspended in RNAse H buffer. This tube is also incubated at 37 C
for 20 minutes, centrifuged and this supernatant is added back to the first digestion tube, the process is repeated several times.
It has been observed that in between such cycling, heating to 65 C before lowering to 37 C
makes the system more efficient. With shorte~_ , oligonucleotide probes such heating is not necessary.
After 10 such repetitions, the supernatant is analyzed for the presence of any RNA or RNA-DNA
conjugate. This is done by monitoring the labels on the RNA or DNA.

Claims (20)

1. A process for amplifying the signal generated from the hybridization of at least one specific nucleic acid sequence in a sample containing a nucleic acid or a mixture of nucleic acids, which process comprises (a) mixing a sample containing at least one specific nucleic acid sequence with a molar excess of RNA which is complementary to said DNA sequence, under hybridization conditions such that a DNA-RNA
hybrid is formed, (b) treating said DNA-RNA hybrid of step (a) with an enzyme capable of digesting said RNA of said DNA-RNA hybrid, to generate low molecular weight unhybridized RNA fragments from said DNA sequence resulting from step (b).

2. The process of claim 1 wherein said RNA of said DNA-RNA hybrid of step (b) is completely digested.

3. The process of claim 1,wherein said enzyme is ribonuclease H.

4. The process of claim 1 wherein said step (b) and (c) are repeated at least once.

5. The process of claim 1, wherein said nucleic acid is double stranded and its strands are separated by physical, chemical, and enzymatic means before or during step (a).

6. The process of claim 1 wherein a molar excess of a second RNA which is complementary to a second DNA sequence in said sample is aded to step (a).

7. The process of claim 1 wherein the process of step (b) can be terminated by increasing the temperature to a level sufficient to inhibit digestion of said RNA of said DNA-RNA hybrid.

8.The process of claim 1, wherein the probe in step (a) is each present in a molar ratio of at least 1000:1 probe:complementary strand.

9. The process of claim l; wherein, due to the degeneracy of the genetic code, a collection of probes are employed for each complementary strand, the sequence of one of which probes is exactly complementary to said complementary strand over the length of the probe.

10. The process of claim 1 further including the step of analyzing the signal generated.

11. The process of claim 10 wherein said low molecular weight RNA fragments are analyzed for identification for the presence or absence of DNA
sequence complemenatry to said RNA.

12. The process of claim 11 wherein said step of analyzing the signal generated includes absorbance, fluoresence, colorometric or radioisotopic.

13. The process of claim 1 wherein said molar excess of RNA which is complementary to said DNA
sequence is immobilized.

14. The process of claim 10 wherein said molar excess of RNA which is complementary to said DNA
sequence is immobilized.

15. The process of claim 1 wherein said DNA is immobilized.

16. The process of claim 10 wherein said DNA is immobilized.

17. The process of claim 13 wherein said immobilized RNA is labeled.

18. The process of claim 15 wherein said immobilized DNA is labeled.

19. The process of claim 13 wherein said nonimmobilized RNA is labeled.

20. The process of claim 16 wherein said immobilized DNA is labeled.