WO1990001068A1 - Sequence specific assay for the detection of a nucleic acid molecule - Google Patents

Sequence specific assay for the detection of a nucleic acid molecule Download PDF

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Publication number
WO1990001068A1
WO1990001068A1 PCT/US1989/002612 US8902612W WO9001068A1 WO 1990001068 A1 WO1990001068 A1 WO 1990001068A1 US 8902612 W US8902612 W US 8902612W WO 9001068 A1 WO9001068 A1 WO 9001068A1
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Prior art keywords
sequence
molecule
nucleic acid
promoter
acid molecule
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PCT/US1989/002612
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French (fr)
Inventor
Mark Berninger
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Life Technologies, Inc.
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/6865Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

Abstract

The invention provides a sensitive means for detecting the presence of a nucleic acid species of desired sequence in a sample. The invention accomplishes this goal through the use of specialized recombinant molecules. The invention pertains to the method for detection, to the recombinant molecules used in the detection method, and to kits which contain the recombinant molecules of the invention.

Description

TITLE OF THE INVENTION

SEQUENCE SPECIFIC ASSAY FOR THE DETECTION OF A NUCLEIC ACID MOLECULE

FIELD OF THE INVENTION:

The present invention relates to a method for detecting the presence of a nucleic acid in a biological sample. The method can be used in med-ical diagnostics, environmental monitoring, or any other use requiring' the detection of specific DNA or RNA' at low concentration.

BACKGROUND OF THE INVENTION

Assays capable of detecting the presence of a particular nucleic acid molecule in a sample are of substantial importance in forensic medicine, in epidemiology and public health, and in the prediction and diagnosis of disease. Such assays can be used, for example, to identify the causal agent of an infectious disease, to predict the likelihood that an individual will suffer from a genetic disease, to determine the purity of drinking water or milk, or to identify tissue samples. The desire to increase the utility and applicability of such assays is often frustrated by assay sensitivity. Hence, it would be highly desirable to develop more sensitive detection assays.

Nucleic acid detection assays can be predicated on any charac¬ teristic of the nucleic acid molecule, such as its size, sequence, and, if DNA, susceptibility to digestion by restriction endonucleases, etc. The sensitivity of such assays may be increased by altering the manner in which detection is reported or signaled to the observer. Thus, for example, assay sensitivity can be increased through the use of detectably labeled reagents. A wide variety of such labels have been used for this purpose. Kourilsky et al . (U.S. Patent 4,581,333) describe the use of enzyme labels to increase sensitivity in a detec¬ tion assay. Radioisotopic labels are disclosed by Falkow et al . (U.S. Patent 4,358,535), and by Berπinger (U.S. Patent 4,446,237). Fluorescent labels (Albarella et al . , EP 144914), chemical labels (Sheldon III et al .. U.S. Patent 4,582,789; Albarella et al .. U.S. Patent 4,563,417), modified bases (Hiyoshi et al . , EP 119448), etc. have also been used in an effort to improve the efficiency with which detection can be observed.

Although the use of highly detectable labeled reagents can improve the sensitivity of nucleic acid detection assays, the sensitivity of such assays remains limited by practical problems largely which are related to non-specific reactions which increase the background signal produced in the absence of the nucleic acid the assay is designed to detect. Thus, for some applications, such as for the identification of a pure culture of a bacteria, the concentration of the desired molecule will typically be amenable to detection, whereas, for other potential applications, the anticipated concentration of the desired nucleic acid molecule will be too low to permit its detection by any of the above-described assays.

One method for overcoming the sensitivity limitation of nucleic acid concentration is to selectively amplify the nucleic acid whose detection is desired prior to performing the assay. Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized. Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al . (U.S. Patent 4,237,224), Maniatis, T., et al . , etc.

Recently, an in vitro, enzymatic method has been described which is capable of increasing the concentration of such desired nucleic acid molecules. This method has been referred to as the "poly erase chain reaction or "PCR" (Mullis, K. et al .. Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al . , EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., US 4,683,202; Erlich, H., US 4,582,788; and Saiki, R. et al .. US 4,683,194).

The polymerase chain reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single- or double-stranded DNA. The essence of the method involves the use of two oligonucleotide probes to serve as primers for the tempia.te-dependent, polymerase mediated replication of a desired nucleic acid molecule.

The precise nature of the two oligonucleotide probes of the PCR method is critical to the success of the method. As is well known, a molecule of DNA or RNA possesses directionality, which is conferred through the 5'-3' linkage of the phosphate groups of the molecule. Sequences of DNA or RNA are linked together through the formation of a phosphodiester bond between the terminal 5' phosphate group of one sequence and the terminal 3' hydroxyl group of a second sequence. Polymerase dependent amplification of a nucleic acid molecule proceeds by the addition of a 5' nucleotide triphosphate to the 3' hydroxyl end of a nucleic acid molecule. Thus, the action of a polymerase extends the 3' end of a nucleic acid molecule. These inherent properties are exploited in the selection of the oligonucleotide probes of the PCR. The oligonucleotide sequences of the probes of the PCR method are selected such that they contain sequences identical to, or complemen¬ tary to, sequences which flank the particular nucleic acid sequence whose amplification is desired. More specifically, the oligonucleo¬ tide sequences of the "first" probe is selected such that it is capable of hybridizing to an o igonucleotide sequence located 3' to the desired sequence, whereas the oligonucleotide sequence of the "second" probe is selected such that it contains an oligonucleotide sequence identical to one present 5' to the desired region. Both probes possess 3' hydroxy groups, and therefore can serve as primers for nucleic acid synthesis.

In the polymerase chain reaction, the reaction conditions are cycled between those conducive to hybridization and nucleic acid polymerization, and those which result in the denaturation of duplex molecules. In the first step of the reaction, the nucleic acids of the sample are transiently heated, and then cooled, in order to denature any double-stranded molecules which may be present. The "first" and "second" probes are then added to the sample at a con¬ centration which greatly exceeds that of the desired nucleic acid molecule. When the sample is incubated under conditions conducive to hybridization and polymerization, the "first" probe will hybridize to the nucleic acid molecule of the sample at a position 3' to the sequence to be amplified. If the nucleic acid molecule of the sample was initially double-stranded, the "second" probe will hybridize to the complementary strand of the nucleic acid molecule at a position 3' to the sequence which is the complement of the sequence whose amplifi¬ cation is desired. Upon addition of a polymerase, the 3' ends of the "first" and (if the nucleic acid molecule was double-stranded) "second" probes will be extended. The extension of the "first" probe will result in the synthesis of an oligonucleotide having the exact sequence of the desired nucleic acid. Extension of the "second" probe will result in the synthesis of an oligonucleotide having the exact sequence of the complement of the desired nucleic acid.

The PCR reaction is capable of exponential amplification of specific nucleic acid sequences because the extension products of the "first" probe contains a sequence which is complementary to a sequence of the "second" probe, and thus will serve as a template for the production of an extension product of the "second" probe. Similarly, the extension products of the "second" probe, of necessity, contain a sequence which is complementary to a sequence of the "first" probe, and thus will serve as a template for the production of an extension product of the "first" probe. Thus, by permitting cycles of polymerization, and denaturation, a geometric increase in the concentration of the desired nucleic acid molecule can be achieved. Reviews of the polymerase chain reaction are provided by Mullis, K.B. (Cold Spring Harbor Sv p. Quant. Biol. 5J.:263-273 (1986)); Saiki, R.K., et al. (Bio/Technology 3:1008-1012 (1985)); and Mullis, K.B., et al. (Met. Enzvmol. 155:335-350 (1987)).

Although the polymerase chain reaction ^provides a method for achieving the amplification of a particular nucleic acid molecule in an unfractionated sample, the method has several disadvantages. First, the method requires the identification and use of two oligo- nucle.otide probes (which are highly specific for the target DNA), and therefore requires a substantial threshold of sequence information before it can be employed. Secondly, the polymerase chain reaction may require a special processing of the sample so the target DNA is in a solution conducive to the enzymatic activity of the DNA polymerase which catalyses the extension of the probe along the target DNA. Thirdly, amplification requires repeated heating and cooling of the reactants to achieve amplification. Fourthly, the method produces an abundance of the same material which it is being used to detect. As a consequence, contamination of the laboratory with the produced DNA can readily occur. Such contamination can result in the generation of "false positive" results, and therefore detract from the overall utility of the method. Care must be taken to achieve successive rounds of denaturation and hybridization. These drawbacks substantially limit the applicability of the polymerase chain reaction to problems of molecular biology, forensic medicine, and diagnostics. Hence, a need exists for a method capable of amplifying the concentration of a particular nucleic acid molecule which (1) does not require the identification of ol igonucleotides which are highly specific for the target sequence, (2) does not require complex sample processing prior to the amplification, (3) does not require repeated cooling and heating of the reactants, and (4) does not result in the production of the same nucleic acid which one is attempting to detect.

Brief Description of the Figures

Figure 1 shows a diagrammatical representation of the recombinant molecule of the first preferred embodiment of the invention.

Figure 2 shows a diagrammatical representation of the recombinant molecules of the second preferred embodiment of the invention.

Summary of the Invention

The invention concerns a method for detecting the presence of a particular desired nucleic acid molecule in a sample. The invention accomplishes this goal through the use of specialized recombinant molecules and kits.

In detail, in one embodiment, the invention provides, a recombinant molecule containing a probe sequence, a promoter sequence and a reporter sequence, wherein the probe sequence is linked to the promoter sequence, and the promoter sequence is operably linked to the reporter sequence.

The invention further provides the above recombinant molecule wherein the promoter and reporter sequences of the molecule are single stranded or wherein the probe sequence of the molecule is hybridized to a complementary target nucleic acid, or wherein the complementary target nucleic acid molecule has a 3' terminus suitable for extension by a polymerase enzyme.

The invention provides the above recombinant molecule wherein the extension of the 3' terminus by a polymerase enzyme results in the for ation of a functional promoter, or wherein the functional promoter is capable of permitting an RNA polymerase to transcribe the operably linked reporter sequence.

The invention also provides a recombinant molecule comprising: a first nucleic acid molecule, the first nucleic acid molecule having a promoter sequence complement linked to the 5' terminus of a probe sequence; and a second, target, nucleic acid molecule whose 3' terminus is hybridized to the probe sequence of the first nucleic acid molecule, and which is of greater, equal, or lesser length than the probe sequence.

The invention also provides the above recombinant molecule wherein the extension of the 3' terminus by a polymerase enzyme results in the formation of a functional promoter, or wherein the functional promoter is capable of. permitting an RNA polymerase to transcribe the second, target, nucleic acid molecule.

The invention further provides a method for detecting a desired target nucleic acid molecule in a sample, the method comprising: a. contacting the target molecule of the sample with the recombinant molecule of claim 1, wherein the nucleotide sequence of the probe sequence of the recombinant molecule is selected to be capable of hybridizing to the target molecule to form a hybridized product, the product having a 3' terminus suitable for extension by a polymerase enzyme; b. incubating the hybridized product in the presence of a polymerase enzyme under conditions sufficient to permit the polymer¬ ase enzyme to extend the 3' terminus to form a double-stranded promoter; c. incubating the hybridized product having the double-stranded promoter in the presence of an RNA polymerase under conditions sufficient to permit the transcription of the reporter sequence; and d. determining the presence of the desired target nucleic acid molecule by assaying for the transcription of the reporter sequence. The invention also provides a method for detecting a target nucleic acid molecule in a sample, the method comprising: a. treating the sample to render the 3'' terminus of the target molecule (i) capable of hybridizing to the probe sequence of the first nucleic acid molecule of claim 9, (ii) capable of enzymatic extension (i.e. having a hydroxyl residue at the 3' position of the ribose or deoxyribose moiety) and (iii) of greater average length than the probe sequence of the first nucleic acid molecule; b. contacting the nucleic acid molecule (a) with the first recombinant molecule to form a hybridized product, the product having a 3' terminus suitable for extension by a polymerase enzyme; c. incubating the hybridized product in the presence of a polymerase enzyme under conditions sufficient to permit the polymer¬ ase enzyme to extend the 3' terminus to form a double-stranded promoter; d. incubating the hybridized product having the double-stranded promoter in the presence of an RNA polymerase under conditions sufficient to permit the transcription of the nucleic acid molecule (a) ; and e. determining the presence of the target nucleic acid molecule by assaying for the transcription of the nucleic acid molecule (a).

The invention also provides kits which are specially adapted to be used in accordance with the above methods.

Description of the Preferred Embodiments

The present invention provides a method for detecting the presence of a particular "target" nucleic acid molecule in a "sample". Such "samples" may include biological samples (such as, for example, blood, stools, sera, urine, saliva, etc.) as well as non-biological samples (such as, for example, waste or drinking water, milk or other foods, air, etc.). The "target" nucleic acid molecules of the present invention may be either DNA or RNA, but is preferably DNA.

One method for detecting such a "target" molecule involves hybridizing a target DNA molecule to be detected to a special DNA probe called a "Probe-Vector" (Life Technologies, Inc.), transforming competent E. col i cells with the target/Probe-Vector hybrid, and observing transfor ants. The process of transformation and the subsequent growth of the transformed bacteria amplify the target DNA one detects by this method. This method is described by Hartley and Berninger (PCT Application Serial No. US84/00525), which reference is herein incorporated by reference in its entirety.

The methods of the present invention permit the detection of such "target" nucleic acid molecules (either DNA or RNA) without requiring the amplification of such molecules. The present invention ac¬ complishes this goal through the construction and use of an assay mixture which contains any of a series of recombinant DNA molecules. The DNA molecules of the present invention contain three sequences of interest: a "probe sequence," a "promoter sequence," and a "reporter sequence complement." In the most preferred embodiments, the recom¬ binant molecules are single-stranded nucleic acid molecules, and most preferably, single-stranded DNA.

The Probe Sequence of the Recombinant Molecules of the Present Invention

The "probe sequence" of the recombinant molecules of the present invention is a nucleic acid molecule whose sequence is capable of identifying the presence of a particular, "target" nucleic acid molecule. The "target" nucleic acid molecule may, for example, be DNA or RNA of viral, viroid, prokaryotic or eukaryotic entities, cells or organisms. Indeed, their are no constraints to the nucleotide sequence, or origin, of the "target" nucleic acid molecule. In one embodiment of the present invention, the nucleotide sequence of the "probe sequence" of the present invention is selected such that it is capable of hybridizing with the "target" nucleic acid molecule. In a second embodiment, the "probe sequence" will have a nucleotide sequence which is substantially identical to the "target" DNA or RNA. In this second embodiment, the "target" molecule is detected indirectly through the detection of a molecule, in the sample, having a complementary sequence. The use of these embodiments is equivalent where the desired molecule is a sequence present on one strand of a double-stranded molecule. As used herein, one nucleotide sequence is said to be "substantially similar" to another nucleotide sequence if the same sequence of nucleotides of a nucleic acid capable of hybridizing to one sequence will also be capable of hybridizing to the other.

The Promoter Sequence and Promoter Sequence Complement of the Recom¬ binant Molecules of the Present Invention

A promoter is a double-stranded DNA or RNA molecule which is capable of binding RNA polymerase and promoting the transcription of an "operably linked" nucleic acid sequence. As used herein, a "promoter sequence" is the sequence of the promoter which is found on that strand of the DNA or RNA which is transcribed by the RNA polymer¬ ase. A "promoter sequence complement" is a nucleic acid molecule whose sequence is the complement of a "promoter sequence." Hence, upon extension of a primer DNA or RNA adjacent to a single-stranded "promoter sequence complement" or, of a "promoter sequence," a double- stranded molecule is created which will contain a functional promoter, if that extension proceeds towards the "promoter sequence" or the "promoter sequence complement." This functional promoter will direct the transcription of a nucleic acid molecule which is operably linked to that strand of the double-stranded molecule which contains the "promoter sequence" (and not that strand of the molecule which contains the "promoter sequence complement").

Certain RNA polymerases exhibit a high specificity for such promoters. The RNA polymerases of the bacteriophages T7, T3, and SP-6 are especially well characterized, and exhibit high promoter specificity. The promoter sequences which are specific for each of these RNA polymerases also direct the polymerase to utilize (i.e. transcribe) only one strand of the two strands of a duplex DNA template. The selection of which strand is transcribed is determined by the orientation of the promoter sequence. This selection determines the direction of transcription since RNA is only polymerized enzymatically by the addition of a nucleotide 5' phosphate to a 3' hydroxyl terminus.

Two sequences of a nucleic acid molecule are said to be "operably linked" when they are linked to each other in a manner which either permits both sequences to be transcribed onto the same RNA transcript, or permits an . RNA transcript, begun in one sequence to be extended into the second sequence. Thus, two sequences, such as a promoter sequence and any other "second" sequence of DNA or RNA are operably linked if transcription commencing in the promoter sequence will produce an RNA transcript of the operably linked second sequence. In order to be "operably linked" it is not necessary that two sequences be immediately adjacent to one another.

Thus, as indicated above, in order to function as a promoter, a promoter sequence must be present as a double-stranded molecule. For the purposes of the present invention, the two strands of a functional promoter sequence are referred to as a "transcript" strand and a "complement" strand. The "transcript" strand is that strand of the duplex which will be transcribed by the RNA polymerase (i.e. which serves as the template for transcription). The "complement" strand is the strand which has a sequence complementary to the "transcript" strand, and which must be present, and hybridized to the "transcript" strand, in order for transcription to occur. Thus, when the "transcript" strand of a promoter sequence is operably linked to a second sequence, hybridization of the "transcript" strand with the "complement" strand, will, in the presence of a polymerase, result in the transcription of the "transcript" strand, and will produce an RNA transcript using the sequence of the "transcript" strand as a template.

The promoter sequences of the present invention may be either prokaryotic, eukaryotic or viral. Suitable promoters are repres¬ sive, or, more preferably, constitutive. Examples of suitable prokaryotic promoters include promoters capable of recognizing the T4 (Malik, S. et al .. J. Biol . Chem. 263:1174-1181 (1984); Rosenberg, A.H. et al.. Gene 59:191-200 (1987); Shinedling, S. et al .. J. Molec. Biol. 195:471-480 (1987); Hu, M. et al .. Gene 42:21-30 (1986)), T3, Sp6, and T7 (Chamberlin, M. et al . , Nature 228:227-231 (1970); Bailey, J.N. et al., Proc. Nat! . Acad. Sci . fU.S.A.) 80:2814-2818 (1983); Davanloo, P. et al .. Proc. Nat! . Acad. Sci. (U.S.A.) 81:2035-2039 (1984)) polymerases; the PR and P[_ promoters of bacteriophage λ (The Bacteriophaqe Lambda, Hershey, A.D., Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1973); Lambda II. Hendrix, R. ., Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1980)); the tr_, recA, heat shock, and lacZ promoters of E. coli; the α-amylase (Ul anen, I., et al .. J. Bacteriol. 162:176-182 (1985)) and the σ-28-specific promoters of B. subtil is (Gil an, M.Z., et al .. Gene 32:11-20 (1984)); the promoters of the bacteriophages of Bacillus (Gryczan, T.J., In: The Molecular Biology of the Bacilli. Academic Press, Inc., NY (1982)); Streptomvces promoters (Ward, J.M., et al ., Mol . Gen. Genet. 203:468-478 (1986)); the jnt promoter of. bacteriophage λ; the bla promoter of the J-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl traπsferase gene of pPR325, etc. Prokaryo¬ tic promoters are reviewed by Glick, B.R., (J. Ind. Microbiol. 1:277- 282 (1987)); Cenatiempo, Y. (Biochimie 68:505-516 (1986)); Watson, J.D. et al . (In: Molecular Biology of the Gene, Fourth Edition, Benjamin Cummins, Menlo Park, CA (1987)); and Gottesman, S. (Ann. Rev. Genet. 18:415-442 (1984)). Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, D., et al . , jL. Mol . APPI . Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 3.:355-365 (1982)); the SV40 early promoter (Benoist, C, et al .. Nature (London) 290:304-310 (1981)); and the yeast ga!4 gene promoter (Johnston, S.A., et al . , Proc. Natl . Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P.A., et al .. Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)). All of the above listed references are incorporated by reference herein.

Strong promoters are the most preferred promoters of the preseTtt invention. Examples of such preferred promoters are those which recognize the T3, SP6 and T7 polymerase promoters; the P[_ promoter of bacteriophage λ; the recA promoter and the .-promoter of the mouse metallothionein I gene. The most preferred promoter is one which is capable of recognizing the T7 polymerase promoter. The sequences of such polymerase recognition sequences are disclosed by Watson, J.D. e_t al . (In: Molecular Biology of the Gene. Fourth Edition, Benjamin Cummins, Menlo Park, CA, (1987)).

The Reporter Sequence and Reporter Sequence Complement of the Recom¬ binant Molecules of the Present Invention

The third sequence of the recombinant DNA or RNA molecules of the present invention is a "reporter sequence complement" which may be composed of any set of nucleotides regardless of sequence. The transcription of the "reporter sequence complement" results in the formation of the "reporter sequence." Preferably, the "reporter sequence complement" will have a length of between 5-1,000 bases and, more preferably, 10-100 bases. The "reporter sequence complement" will, preferably, be selected such that the "reporter sequence" will have a physical characteristic such as size, suitability to cleavage by restriction endonuclease, sequence, etc., which will permit or facilitate its identification or detection by means well known in the art. Alternatively, the reporter sequence can contain modified bases which may be used to facilitate its detection. Preferably, the presence of the reporter sequence will be detected through the use of an antibody or an antibody fragment, capable of specific binding to either the reporter sequence, the complement of the reporter sequence or a double-stranded nucleic acid molecule composed of the reporter sequence complement and the reporter sequence.

1

The above-described recombinant molecules can be produced through any of a variety of means, such as, for example, DNA or RNA synthesis, or more preferably, by application of recombinant DNA techniques. Techniques for synthesizing such molecules are disclosed by, for example, Wu, R., et al . (Prog. Nucl . Acid. Res. Molec. Biol. 21:101- 141 (1978)). Procedures for constructing recombinant molecules in accordance with the above-described method are disclosed by Maniatis, T., et al . , In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1984), which reference is herein incorporated by reference.

The 3' terminus of the above-described recombinant molecule is preferably treated to render it unsuitable for enzymatic extension or as an initiation site for RNA polymerase-mediated transcription. Such treatment may be accomplished by blocking the terminus by chemical means, or by modifying the terminal bases such that they sterically interfere with polymerase action. In a preferred embodiment, such treatment is accomplished by immobilizing the 3' terminus, such as by coupling it to a solid support (such as, for example, glass, plastic, latex, etc.). The support may be of any form (i.e. a sheet, rod, sphere, ovoid, etc. Procedures for such immobilization are well known to those of ordinary skill. In a preferred embodiment, the 3' end of the recombinant molecule is covalently bound to the solid support, preferably to paramagnetic microbeads of approximately 4 μm in diameter. A spacer region may be used to extend the probe outward from the solid support as long as (1) it will not sterically hinder any function or characteristic of the recombinant molecule, and (2) the sequence of the spacer region does not participate in the hybridization or polymerization reactions of the assay. It is typically desirable to immobilize several, and preferably, a large number of such recombinant molecule to the support.

The recombinant molecules of the present invention are used in an assay to reveal the presence of extremely minute amounts of a specif¬ ic, desired, DNA or RNA in a sample. The assay reveals the presence of the desired molecule by causing the above-described reporter sequence to be amplified. In order to achieve the amplification of the reporter sequence, the assay requires the addition of a recom¬ binant molecule of the present invention, an amount of sample suspec¬ ted of containing the desired molecule, and at least two polymerases, including both a DNA polymerase (such as E. col i DNA polymerase (and especially the "Klenow" fraction of this polymerase), T4, or T7 DNA polymerase, or another polymerase having similar activity), and an RNA polymerase (such as E. col i RNA polymerase, T3, SP6 or T7 polymerase, or another enzyme having similar activity). The most preferred embodiment employs the "Klenow" fraction of E. col i DNA polymerase, and the RNA polymerase of T7. In addition to the above-described components, it is necessary to provide to the assay mixture an amount of required salts, ATP, CTP, GTP, UTP, dATP, dTTP, dGTP, dCTP, or other nucleotide triphosphates, etc., sufficient to (1) permit nucleic acid polymerization (either both DNA or RNA if both types of polymer¬ ases have been provided, or only DNA or RNA, if only one type of polymerase has been provided) and (2) to render such polymerization non-substrate limited.

The Preferred Recombinant Molecules of the Invention and Their Use

The First Preferred Embodiment

In the first preferred embodiment of the present invention, the recombinant molecule of the invention is a single-stranded DNA molecule composed of a probe sequence whose 5' end is linked to the 3' end of the "transcript" strand of a promoter sequence. The 5' end - of the "transcript" strand of the promoter sequence is operably linked to a reporter sequence complement. Using such a construct, transcrip¬ tion from the promoter sequence (which will require the presence and hybridization of a "complement" sequence) will cause the transcription of the reporter sequence complement. No transcription of the probe sequence will occur.

In the above-described recombinant molecule the promoter sequence is one which preferably recognizes the T7 RNA polymerase. The nucleotide sequence of the probe sequence of the recombinant molecule has been selected such that it is capable of hybridizing to a particular desired "target" nucleic acid molecule, whose detection is sought. The recombinant molecule is immobilized by binding its 3' end to a solid support (preferably through the use of an intervening spacer region). A sample suspected of containing the desired "target" nucleic acid molecule is prepared • for assay by incubating it under conditions capable of fragmenting the nucleic acid molecules of the sample. Such fragmentation can be accomplished by shearing the nucleic acid, subjecting it to x-rays, or other radiation, or by degrading it with an endonuclease. Since the preferred fragments are those which have a 3' hydroxyl end, one preferred treatment involves incubation with one, or several restriction endonucleases which produce cleavage products having 3' hydroxyl ends. A second preferred treatment involves the use of chiral metal complexes (such as those containing Cu, Fe, or Pb) to cleave DNA into fragments having the desired 3' hydroxyl ends (Barton, J., Abstr. Pap. Amer. Chem. Soc. l_94:Biol 85 (1987); Kuwahara, J. et al .. Biochem. 25:1216-1221 (1986); Sugiura, Y. et al .. Biochi . Biophvs. Acta 782:254-261 (1984)). Once the sample has been fragmentized, it is incubated in a manner sufficient to denature any double-stranded nucleic acid which may be present. Typically such a goal can be realized by transiently heating the sample. Once the sample has been treated in the manner described above, a suitable amount of the sample (i.e. an amount suspected to contain at least one molecule having the sequence of the desired "target" molecule) is incubated in the presence of the above-described preferred recombinant molecule under conditions suitable to permit hybridization. An effective amount of T7 RNA polymerase, "Klenow" fragment of E. coli DNA polymerase, and the salts and nucleotide triphosphates required for RNA and DNA synthesis are added. As used herein, an "effective amount" of such compounds is an amount sufficient to render the rate of polymerization (catalyzed by the enzyme) non-enzyme, and non-substrate, limited. Lesser amounts of such compounds may be sufficient to allow the overall process to occur at an acceptable efficiency.

If the sample contains the desired nucleic acid molecule, then the molecule will hybridize to the probe sequence of the recombinant molecule. Since the recombinant molecule of the preferred embodiment possesses additional sequence (such as the promoter and reporter sequence complement) 5' to the probe sequence, any desired "target" nucleic acid molecule which hybridizes to it will form a partial duplex molecule having a recessed 3' hydroxyl end. As will readily be appreciated, this 3' hydroxyl end is the 3' end of the desired "target" nucleic acid molecule. Such a molecule is a substrate for DNA polymerase, and will therefore have its 3' end extended by the DNA polymerase in a direction toward the promoter sequence of the recombinant molecule. This extension shall, of course, be template directed, and will therefore result in the synthesis of an elongated molecule containing a sequence complementary to that of the promoter sequence of the recombinant molecule.

When the 3' end of the desired molecule has been extended to an extent that it now includes a sequence complementary to that of the promoter sequence of the recombinant molecule, a functional, double- stranded, promoter sequence will be formed. This promoter sequence will be recognized by the T7 RNA polymerase of the assay, and will result in the transcription of the reporter sequence complement of the recombinant molecule (which is operably linked to the promoter sequence) .

Repeated transcription of the reporter sequence complement will occur since the double-stranded promoter region is stable under the conditions of the assay, and because of the non-rate limiting con¬ centration of the RNA polymerase, and polymerization substrates. Transcription can be permitted to continue for as long as desired, in order to amplify the concentration of the reporter sequence until it has reached a detectable concentration. Since the reporter molecule will not be synthesized if the desired "target" molecule of the sample (which hybridized to the probe sequence) is absent, the presence of the reporter molecule is indicative of the pVesence of the desired "target" molecule in the sample.

It is to be noted that the concentration of probe molecules will, in almost all instances vastly exceed the concentration of the "target" molecule which one is detecting. These probe molecules contain sequences complementary not only to the "target" being detected, but also to the "reporter sequence complement" synthesized by the RNA polymerase. Since most probe molecules will not hybridize to "target" molecules (too few "target" molecules being present), most probe molecules remain single-stranded and therefore capable of hybridizing to the "reporter sequence" molecules.

The Second Preferred Embodiment

In the second preferred embodiment, the detection of the desired * nucleic acid molecule of the sample is also achieved through the detection of a reporter sequence. The second embodiment of the invention accomplishes such detection through the use of two recom¬ binant molecules.

The first recombinant molecule of this embodiment of the present invention is a single-stranded nucleic acid molecule, most preferably DNA. The molecule contains a "probe sequence" whose 5' end is operably linked to a "promoter sequence complement." Importantly, in this embodiment, the nucleotide sequence of the reporter sequence (discussed in detail below) must be a sequence which is naturally linked to the 5' end of the desired sequence. It is to be noted that, in contrast to the first embodiment of the present invention,, the reporter sequence of the second embodiment is provided by the sample being assayed by the method. A preferred "promoter sequence complement" is the promoter sequence complement of promoters which recognize T7 RNA polymerase. The 5' end of the "probe sequence" is, preferably, immobilized to a solid support (preferably through the use of an intervening spacer region, and most preferably to paramagnetic microbeads of approximately 4 μ in diameter). The recombinant molecule does not preferably contain a reporter sequence or reporter sequence complement.

As in the first embodiment of the invention, the presence of a desired molecule is revealed by the amplification of a reporter sequence. The enzymes, salts, and nucleotides of the above-discussed first embodiment of the invention are also employed for this second embodiment.

The second embodiment of the invention reveals the presence of a desired molecule in the following manner. In the initial step of the second embodiment, the nucleic acid molecules of the sample, suspected of containing the desired "target" nucleic acid molecule (preferably DNA) is incubated (in the same manner employed to prepare the sample of the first embodiment of the invention) in order to produce fragment molecules of random size having 3' hydroxyl ends. The size of the probe sequence of the first recombinant molecule of this embodiment is selected to be shorter than the average size of the fragments of the sample nucleic acids. Through such considerations, it is possible to produce fragments which contain the desired "target" sequence, and among which a substantial number have the following characteristics: 1) they are capable of hybridizing to the probe sequence; 2) the 5' terminus of the fragment extends beyond the 3' terminus of the probe sequence; and 3) the 3' terminus of the fragment does not extend beyond the 5' terminus of the probe sequence. After such treatment, the sample is incubated under conditions sufficient to denature any double-stranded molecules which may be present in the sample.

Once the above-described steps have been completed, the nucleic acid molecules of the sample are incubated, under conditions conducive to hybridization and DNA or RNA polymerization, in the presence of the first probe of this embodiment, and the enzymes and co-factors discussed above.

If the desired "target" nucleic acid molecule is present in the sample, it will be present on one or more fragments of random size, many of which will have a 3' hydroxyl end. Thus, when the fragments of the sample are incubated in the presence of the first recombinant molecule those fragments which contain the desired "target" sequence will hybridize to the probe sequence of the recombinant molecule. Such hybridization will produce a duplex molecule having a recessed 3' hydroxyl end, and thus will create a substrate for DNA polymerase. Importantly, this 3' hydroxyl end is the 3' end of the DNA fragment which contains the desired "target" sequence. Thus, since the reaction mixture contains a DNA polymerase and deoxyribonucleotides, the template directed extension of the hybridized sample fragment will occur. Such extension does not result in the replication of the desired "target" sequence, but rather causes its extension toward the promoter sequence complement of the recombinant molecule.

Extension of the 3' end of the fragment through the "promoter sequence complement" results in the synthesis of a "promoter se¬ quence" operably linked to the sample fragment. Importantly, such extension thereby creates a functional promoter. Since the reaction mixture contains an RNA polymerase (preferably T7 RNA polymerase) and ribonucleotides, transcription of the fragment. No transcription of the recombinant molecule occurs since the recombinant molecule is 1 inked to the "promoter sequence complement" and not to the "promoter sequence. "

Note that the 3' hydroxyl terminus of the probe molecule will be extended along the target DNA to render the reporter sequence region of the target DNA double-stranded.

Since the size of the probe sequence has been selected to be shorter than the average size of the fragments of the sample, tran¬ scription of the fragment shall result in the amplification of not only the complement of the desired "target" molecule, but will also lead to the amplification of that sequence of the fragment which is linked to the 5' end of the desired "target" sequence. This amplified 5' sequence is the reporter sequence of the second embodiment of the invention.

The formation of the reporter sequence is, therefore, dependent upon the initial presence, in the sample, of a fragment which con¬ tained the desired "target" sequence. The presence of the reporter molecule .is detected through the use of the second recombinant molecule of this embodiment. The second recombinant molecule of this embodiment of the present invention is a single-stranded nucleic acid molecule, most preferably DNA. The molecule may have any sequence but must contain a "reporter sequence complement." The 5' end of this recombinant molecule is, preferably, immobilized to a solid support (most preferably through the use of an intervening spacer region). The formation of the reporter molecule can be detected by its ability to hybridize with this second recombinant molecule.

Uses of the Recombinant Molecules of the Invention

The recombinant molecules of the present invention may be adapted to permit their use in identifying or detecting the presence of any desired nucleic acid molecule. These properties render the assays of the present invention suitable for applications in medical diagnostics, environmental monitoring, or any other use requiring the detection of specific DNA or RNA at low concentration.

The assays of the present invention have substantial utility in the fields of epidemiology, food science and waste management. For example, samples of air, water or food (such as milk, dairy products, meat, poultry, etc.) can be incubated in accordance with the methods of the present invention in order to assess and identify the presence of pathogenic bacteria (such as S. typhosa, M. tuberculosi, etc.), yeasts, protozoa, nematodes (such as the causal agent of heartworm, trichinosis, malaria, etc. ) or viruses (such as those responsible for hepatitis, influenza, shipping fever, etc.). For such purposes the recombinant molecules will contain probe sequences complementary to characteristic sequences of such pathogens.

As indicated above, the use of the molecules and assays of the present invention is especially facilitated and enhanced by the use of "kits," whose components are especially adapted to be used with one another. Such kits will typically, contain at least one of *the recom¬ binant molecules of the present invention, and will preferably contain all of the recombinant molecules required to pursue a particular assay. The "probe sequence" of such recombinant molecules will, of course, vary depending upon the intended use. Thus for example, a "kit" to designed to detect papillovirus will contain a recombinant molecule whose probe sequence is complementary to a papillovirus sequence. Likewise, similar kits can be prepared in which the "probe sequence" of the recombinant molecule is complementary to HIV (to detect HIV), prokaryotic RNA (to detect bacterial contamination of foods, or "to detect infection), yeast DNA or RNA (to detect yeast infection), etc.

The kits of the present invention will, preferably, also contain enzymes (such as DNA or RNA polymerases, etc.), nucleotide triphos- phates, salts, etc. The kits may also contain instructional material and brochures describing the mode of preferred use. Although the invention has been primarily described as providing a means for detecting a desired nucleic acid molecule, the invention also contemplates the characterization of such detected molecules. The amplified molecules obtained through the use of the second preferred embodiment of the invention can be analyzed through the use of any of a variety of methods well known in the art in order to further characterize their sequence or nature. For example, such amplified molecules can be sequenced, converted to cDNA using reverse transcriptase, and then cloned, or hybridized against a reference nucleic acid molecule. Such information can be used in diagnostics, and for other uses.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A recombinant molecule containing a probe sequence, a promoter sequence and a reporter sequence, wherein said probe sequence is linked to said promoter sequence, and said promoter sequence is operably linked to said reporter sequence.
2. The recombinant molecule of claim 1 which is single-stranded.
3. The recombinant molecule of claim 1, wherein said promoter and reporter sequences of said molecule are single stranded and wherein said probe sequence of said molecule is hybridized to a complementary target nucleic acid.
4. The recombinant molecule of claim 3 wherein said complementary target nucleic acid molecule has a 3' terminus suitable for extension by a polymerase enzyme.
5. The recombinant molecule of claim 4 wherein the extension of said 3Λ terminus by a polymerase enzyme results in the formation of a functional promoter.
6. The recombinant molecule of claim 5 wherein said functional promoter is capable of permitting an RNA polymerase to transcribe said operably linked reporter sequence.
7. The recombinant molecule of claim 1 wherein said promoter sequence is a promoter sequence is substantially similar to a promoter sequence of T4, T7, or lambda.
8. The recombinant molecule of claim 1 wherein said molecule is immobilized onto a solid support.
9. A recombinant molecule comprising: a first nucleic acid molecule, said first nucleic acid molecule having a promoter sequence complement linked to the 5' terminus of a probe sequence; and a second, target, nucleic acid molecule whose 3' terminus is hybridized to the probe sequence of said first nucleic acid molecule, and which is of greater length than said probe sequence.
10. The recombinant molecule of claim 9 wherein the extension of said 3' terminus by a polymerase enzyme results in the formation of a functional promoter.
11. The recombinant molecule of claim 10 wherein said functional promoter is capable of permitting an RNA polymerase to transcribe said second, target, nucleic acid molecule.
12. The recombinant molecule of claim 10 wherein said promoter has a sequence which is substantially similar to a promoter sequence of T4, T7, or lambda.
13. The recombinant molecule of claim 9 wherein said molecule is immobilized onto a solid support.
14. A method for detecting a desired target nucleic acid molecule in a sample, said method comprising: a. contacting said target molecule of said sample with the recombinant molecule of claim 1, wherein the nucleotide sequence of said probe sequence of said recombinant molecule is selected to be capable of hybridizing to said target molecule to form a hybridized product, said product having a 3' terminus suitable for extension by a polymerase enzyme; b. incubating said hybridized product in the presence of a polymerase enzyme under conditions sufficient to permit said polymer- ase enzyme to extend said 3' terminus to form a double-stranded promoter; c. incubating said hybridized product having said double- stranded promoter in the presence of an RNA polymerase under condi¬ tions sufficient to permit the transcription of said reporter se¬ quence; and d. determining the presence of said desired target nucleic acid molecule by assaying for the transcription of said reporter sequence.
15. A method for detecting a target nucleic acid molecule in a sample, said method comprising: a. treating said sample to render the 3' terminus of said target nucleic acid molecule (i) capable of hybridizing to the probe sequence of said first nucleic acid molecule of claim 9, and (ii) of greater length than said probe sequence of said first nucleic acid molecule; b. contacting said nucleic acid molecule (a) with said, first recombinant molecule to form a hybridized product, said product having a 3' terminus suitable for extension by a polymerase enzyme; c. incubating said hybridized product in the presence of a polymerase enzyme under conditions sufficient to permit said polymer¬ ase enzyme to extend said 3' terminus to form a double-stranded promoter; d. incubating said hybridized product having said double- stranded promoter in the presence of an RNA polymerase under condi¬ tions sufficient to permit the transcription of said nucleic acid molecule (a); and e. determining the presence of said desired target nucleic acid molecule by assaying for the transcription of said nucleic acid molecule (a).
16. A kit being specialized to contain in close confinement: a recombinant molecule, said molecule selected from the group cons sting of the recombinant molecule of claim 1 or the first recombinant molecule of claim 9; and an RNA polymerase, wherein said polymerase is capable of binding to the promoter sequence and promoter sequence complement of said recombinant molecule.
17. The kit of claim 16 which additionally includes a DNA polymerase enzyme.
PCT/US1989/002612 1988-07-22 1989-06-15 Sequence specific assay for the detection of a nucleic acid molecule WO1990001068A1 (en)

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US6100024A (en) * 1991-02-08 2000-08-08 Promega Corporation Methods and compositions for nucleic acid detection by target extension and probe amplification
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