CA1340807C - Nucleic acid amplification process - Google Patents
Nucleic acid amplification processInfo
- Publication number
- CA1340807C CA1340807C CA000559709A CA559709A CA1340807C CA 1340807 C CA1340807 C CA 1340807C CA 000559709 A CA000559709 A CA 000559709A CA 559709 A CA559709 A CA 559709A CA 1340807 C CA1340807 C CA 1340807C
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- Prior art keywords
- dna
- rna
- template
- polymerase
- process according
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/702—Specific hybridization probes for retroviruses
- C12Q1/703—Viruses associated with AIDS
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
This invention relates to a process for amplifying a specific nucleic acid sequence. The process involves synthesizing single-stranded RNA, single-stranded DNA and double-stranded DNA. The single-stranded RNA is a first template for a first primer, the single-stranded DNA is a second template for a second primer, and the double stranded DNA is a third template for synthesis of a plurality of copies of the first template. A sequence of the first primer or the second primer is complementary to a sequence of the specific nucleic acid and a sequence of the first primer or the second primer is homologous to a sequence of the specific nucleic acid. The amplification process may be used to increase the quantity of the specific nucleic acid sequence to allow detection, or to increase the purity of the specific nucleic acid sequence as a substitute for conventional cloning methodology.
Description
NUCLEIC ACID AMPLIFICATION PROCESS
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FIELD OF THE INVENTION
This invention relates to a process for amplifying a specific nucleic acid sequence.
BACKGROUND OF THE INVENTION
The detection of a specific nucleic acid sequence present in a sample by probing the sample with a complementary sequence of nucleic acids is a known diagnostic technique. Nucleic acids are highly specific in binding to complementary nucleic acids and are thus useful to determine whether a specific nucleic acid is present in a sample. One must know the sequence of the specific nucleic acid to be detected and then construct a probe having a complementary nucleic acid sequence to the specific nucleic acid sequence.
In this application, the phrase "specific nucleic acid sequence" means a single stranded or double ,_ ..
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stranded nucleic acid; "sample" means a mixture containing nucleic acids; "sufficiently complementary" means that two nucleic acids, a primer and a template, are capable of specific interaction which allows efficient, primer-dependent and template-directed synthesis of DNA, under given conditions of ionic strength and temperature; and "sufficiently homologous"
means that two nucleic acids have similar DNA
sequences and are each capable of interacting with the same DNA primer.
Since nucleic acid probes are highly specific, it is preferable in some situations to probe the nucleic acid sequence itself rather than the protein produced by the nucleic acid sequence. As a particular example, a diagnostic method based solely on protein detection would be unreliable for determining the presence of infectious particles of hepatitis B virus, due to the presence of significant levels of non-infectious antigen particles which lack the DNA
genome. In another example, the various subtypes of human papilloma virus found in either pre-cancerous or benign cervical tumors can be distinguished only by the use of nucleic acid probe hybridization. Also, the microbiology of AIDS makes it certain that an assay based on the presence of AIDS specific nucleic acid sequence would be superior as a diagnostic.
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FIELD OF THE INVENTION
This invention relates to a process for amplifying a specific nucleic acid sequence.
BACKGROUND OF THE INVENTION
The detection of a specific nucleic acid sequence present in a sample by probing the sample with a complementary sequence of nucleic acids is a known diagnostic technique. Nucleic acids are highly specific in binding to complementary nucleic acids and are thus useful to determine whether a specific nucleic acid is present in a sample. One must know the sequence of the specific nucleic acid to be detected and then construct a probe having a complementary nucleic acid sequence to the specific nucleic acid sequence.
In this application, the phrase "specific nucleic acid sequence" means a single stranded or double ,_ ..
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stranded nucleic acid; "sample" means a mixture containing nucleic acids; "sufficiently complementary" means that two nucleic acids, a primer and a template, are capable of specific interaction which allows efficient, primer-dependent and template-directed synthesis of DNA, under given conditions of ionic strength and temperature; and "sufficiently homologous"
means that two nucleic acids have similar DNA
sequences and are each capable of interacting with the same DNA primer.
Since nucleic acid probes are highly specific, it is preferable in some situations to probe the nucleic acid sequence itself rather than the protein produced by the nucleic acid sequence. As a particular example, a diagnostic method based solely on protein detection would be unreliable for determining the presence of infectious particles of hepatitis B virus, due to the presence of significant levels of non-infectious antigen particles which lack the DNA
genome. In another example, the various subtypes of human papilloma virus found in either pre-cancerous or benign cervical tumors can be distinguished only by the use of nucleic acid probe hybridization. Also, the microbiology of AIDS makes it certain that an assay based on the presence of AIDS specific nucleic acid sequence would be superior as a diagnostic.
x The greatest difficulty with applying exis~~ ~~
nucleic acid probe technology, and the reason that the utility of the existing probe technology is limited, is the copy number problem. In a virus or cell, for example, there is usually a single copy of a particular gene. This one copy may give rise to many copies of gene product, either RNA or protein. For this reason, diagnostic techniques have often involved probing the protein, since the specific sequence of nucleic acid to be detected may give rise to many thousand copies of protein.
The naturally-occurring high number of ribosomal RNA, up to 100,000 copies per cell, has been used by GenProbe to facilitate diagnosis of certain bacterial pathogens, such as Legionella and Mycoplasma, using nucleic acid probes. However, this strategy cannot be used with non-cellular pathogens, such as viruses.
Copy number is a particular problem with the development of a nucleic acid probe method for the detection of AIDS virus, where the integrated provirus may be present in less than one of ten thousand peripheral blood lymphocytes. Thus, if the particular nucleic acid sequence suspected to be present in a sample could be amplified, the copy number problem could be circumvented and probe assays could be more readily used.
nucleic acid probe technology, and the reason that the utility of the existing probe technology is limited, is the copy number problem. In a virus or cell, for example, there is usually a single copy of a particular gene. This one copy may give rise to many copies of gene product, either RNA or protein. For this reason, diagnostic techniques have often involved probing the protein, since the specific sequence of nucleic acid to be detected may give rise to many thousand copies of protein.
The naturally-occurring high number of ribosomal RNA, up to 100,000 copies per cell, has been used by GenProbe to facilitate diagnosis of certain bacterial pathogens, such as Legionella and Mycoplasma, using nucleic acid probes. However, this strategy cannot be used with non-cellular pathogens, such as viruses.
Copy number is a particular problem with the development of a nucleic acid probe method for the detection of AIDS virus, where the integrated provirus may be present in less than one of ten thousand peripheral blood lymphocytes. Thus, if the particular nucleic acid sequence suspected to be present in a sample could be amplified, the copy number problem could be circumvented and probe assays could be more readily used.
In a normal biological sample, containing only a few cells, and consequently only a few copies of a particular gene, it is necessary to utilize an amplification process in order to overcome the copy number problem.
One method to amplify is to 'grow out' the sample, that is, to arrange conditions so that the living biological material.present in the sample can replicate itself. Replication increases the quantity of nucleic acid sequences to detectable levels. In the food industry, for example, in order to test processed food for the food-poisoning bacteria Salmonella, food samples must be incubated for a number of days to increase the quantity of nucleic acids. In clinical samples, pathogens must also be allowed to increase their number by growing out over some considerable time.
United States Patent No. 4,683,195 issued on July 28. 1987 to Cetus Corporation and United States Patent No. 4,683,202 issued on July 28, 1987 to Cetus Corporation are each directed to a process for amplifying a target nucleic acid sequence contained in a sample. United States Patent No. 4,683,195 relates ".r. ~. .
1340~~7 to a process in which a sample suspected of containing a target nucleic acid sequence is treated with oligonucleotide primers such that a primer extension product is synthesized which in turn serves as a template, resulting in amplification of the target nucleic acid sequence. The primer extension product is separated from the template in the preferred embodiment using heat denaturation. Similarly, United States Patent No. 4,683.202 relates to a process for amplifying a target nucleic acid sequence having two separate complementary strands. The process includes treating the strands with primers to synthesize extension products, separating the primer extension products from the templates, and in turn using the primer extension products as templates.
Both of the above United States patents require either manual or mechanical participation and multi-step operations by the user in the amplification process. The steps involved in these patents require the user to heat the sample, cool the sample, add appropriate enzymes and then repeat the steps. The temperature changes cause the enzymes to lose their activity. Hence, the user is required to repeatedly supplement the amplification mixture with aliquots of appropriate enzymes during the amplification process.
One method to amplify is to 'grow out' the sample, that is, to arrange conditions so that the living biological material.present in the sample can replicate itself. Replication increases the quantity of nucleic acid sequences to detectable levels. In the food industry, for example, in order to test processed food for the food-poisoning bacteria Salmonella, food samples must be incubated for a number of days to increase the quantity of nucleic acids. In clinical samples, pathogens must also be allowed to increase their number by growing out over some considerable time.
United States Patent No. 4,683,195 issued on July 28. 1987 to Cetus Corporation and United States Patent No. 4,683,202 issued on July 28, 1987 to Cetus Corporation are each directed to a process for amplifying a target nucleic acid sequence contained in a sample. United States Patent No. 4,683,195 relates ".r. ~. .
1340~~7 to a process in which a sample suspected of containing a target nucleic acid sequence is treated with oligonucleotide primers such that a primer extension product is synthesized which in turn serves as a template, resulting in amplification of the target nucleic acid sequence. The primer extension product is separated from the template in the preferred embodiment using heat denaturation. Similarly, United States Patent No. 4,683.202 relates to a process for amplifying a target nucleic acid sequence having two separate complementary strands. The process includes treating the strands with primers to synthesize extension products, separating the primer extension products from the templates, and in turn using the primer extension products as templates.
Both of the above United States patents require either manual or mechanical participation and multi-step operations by the user in the amplification process. The steps involved in these patents require the user to heat the sample, cool the sample, add appropriate enzymes and then repeat the steps. The temperature changes cause the enzymes to lose their activity. Hence, the user is required to repeatedly supplement the amplification mixture with aliquots of appropriate enzymes during the amplification process.
In addition, in United States Patents Nos. 4,683,195 and 4,683,202 each cycle of the amplification process takes place by the synthesis from a first template, of a second template, the second template in turn is used to synthesize the first template. This procedure is repeated, thus, each cycle of the amplification process is based on the synthesis of one product from one substrate.
Notwithstanding the amplification processes disclosed in the prior art, a need exists for improvements to the amplification process. It would be preferable if the amplification process required less participation and fewer manipulations by the user. Further, it would be advantageous if the amplification took place at a relatively constant ambient temperature so that the activity of the enzymes involved in the process would not be affected. It would be more expedient if a template could be used to generate more than one product from one substrate in each cycle of the amplification process.
Notwithstanding the amplification processes disclosed in the prior art, a need exists for improvements to the amplification process. It would be preferable if the amplification process required less participation and fewer manipulations by the user. Further, it would be advantageous if the amplification took place at a relatively constant ambient temperature so that the activity of the enzymes involved in the process would not be affected. It would be more expedient if a template could be used to generate more than one product from one substrate in each cycle of the amplification process.
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SUN~1ARY OF THE INVENTION
This invention relates to an amplification process which is expedient and requires less participation and fewer manipulations by the user of the process than conventional amplification processes. The amplification takes place at a relatively constant ambient temperature. In addition, each cycle of the process generates a plurality of copies of product from one substrate. The amplification process of this invention may be used to increase the quantity of a specific nucleic acid thus circumventing the copy number problem. Hence, probe assays may be more readily used. The amplification process could also be used to increase the purity of a specific nucleic acid sequence as a substitute for conventional cloning methodology.
One aspect of the invention is a process for amplifying a specific nucleic acid sequence. The process involves the synthesis of single-stranded RNA, single-stranded DNA, and double stranded DNA. The single stranded RNA is a first template for a first primer. The single stranded DNA is a second template for a second ~3~O~u7 primer. The double stranded DNA is a third template for the synthesis of a plurality of copies of the first template. A sequence of the first or the second primer is sufficiently complementary to a sequence of the specific nucleic acid sequence and a sequence of the first or the second primer is sufficiently homologous to a sequence of the specific nucleic acid sequence. A 3' end of the first primer is oriented towards a 3' end of the second primer on complementary strands.
Another aspect of the invention is a process for amplifying a specific nucleic acid sequence. The process involves:
(a) hybridizing a first primer to a first template. The first primer has a DNA
sequence which is sufficiently complementary to a RNA sequence of the first template;
(b) synthesizing a first DNA sequence covalently attached to the first primer and complementary to the RNA sequence of the first template. The first DNA sequence and the first primer comprise a second template;
_ g _ (c) separating the first template from the second template to allow hybridization of a second primer;
(d) hybridizing the second primer to the second template. The second primer has a DNA
sequence which is sufficiently complementary to a DNA sequence of the second template.
(e) synthesizing a second DNA sequence covalently attached to the second primer and complementary to the DNA sequence of the second template and synthesizing a third DNA
sequence covalently attached to the second template and complementary to the DNA
sequence of the second primer. The second and third DNA sequences, the second primer and the second template comprise a third template;
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- to -(f~ synthesizing a plurality of copies of the RNA sequence of the first template from the third template.
A sequence of the first or the second primer is sufficiently complementary to a sequence of the specific nucleic acid sequence and a sequence of the first or the second primer is sufficiently homologous to a sequence of the specific nucleic acid sequence.
A 3' end of the first primer is oriented towards a 3' end of the second primer on complementary strands.
In a further alternative of the invention, the second primer of DNA has a sequence at its 3' end which is sufficiently complementary to the DNA
sequence of the second template. The second primer has at its 5' end an antisense sequence of a promoter and an antisense sequence of a transcription initiation site for a RNA polymerase.
13~08i37 Another alternative of the invention is a process for amplifying a specific nucleic acid sequence. The process involves combining a first primer, a second primer, ribonuclease H, a RNA-directed DNA polymerase, a DNA-directed DNA polymerase, a RNA polymerase, ribonucleoside triphosphates and deosyribonucleoside triphosphates with a sample. The first primer of DNA
has a sequence which is sufficiently complementary to a first template of RNA. The second primer of DNA has a sequence which is sufficiently complementary to a second template of DNA, and an antisense sequence of a promoter and an antisense sequence of a transcription initiation site which are recognized as substrate by the RNA polymerase. A sequence of the first primer or the second primer is sufficiently complementary to a sequence of the specific nucleic acid sequence and a sequence of the first primer or the second primer is sufficiently homologous to a sequence of the specific nucleic acid. A 3' end of the first primer is oriented towards a 3' end of the second primer on complementary strands.
A further alternative of the invention is a process for amplifying a specific nucleic acid sequence. The process involves adding a first .,.....
primer, a second primer, avian myeloblastosis viral polymerase, E. coli ribonuclease H, bacteriophage T7 RNA polymerase, ribonucleoside triphosphates and deoxyribonucleoside triphosphates to a sample. The first primer of DNA has a sequence which is sufficiently complementary to a first template of RNA. The second primer of DNA has a sequence which is sufficiently complementary to a second template of DNA, and an antisense sequence of a promoter and an antisense sequence of a transcription initiation site which are recognized as substrate by T7 RNA polymerase. A sequence of the first primer or the second primer is sufficiently complementary to a sequence of the specific nucleic acid sequence and a sequence of the first primer or the second primer is sufficiently homologous to a sequence of the specific nucleic acid sequence. A 3' end of the first primer is oriented towards a 3' end of the second primer on complementary strands.
BRIEF DESCRIPTION OF THE DRAWINGS
In drawings which illustrate embodiments of the invention, x 134080' Figure 1 is a general illustration of the nucleic acid amplification process;
Figure 2 shows the synthetic oligonucleotide DNA
sequences which are used for testing the amplification process: Figure 2A, the gag test sequence; Figure 2B, the gag2 test sequence;
Figure 3 is an autoradiogram of PAGE analysis of amplification reactions using different primer concentrations;
Figure 4 is an autoradiogram of PAGE analysis of amplification reactions using different template concentrations;
Figure 5 is an autoradiogram of Dot-blot hybridization on amplification reactions;
Figure 6 is an autoradiogram of PAGE analysis of amplification reaction using restriction fragments as template;
1340~~'7 Figure 7 is a general illustration of the indirect nucleic acid amplification process.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
This invention relates to a process for amplifying a specific nucleic acid sequence. The amplification involves the alternate synthesis of DNA
and RNA and is generally illustrated in Figure 1. In this process. single-stranded RNA is converted to single-stranded DNA which in turn is converted to a functional template for the synthesis of a plurality of copies of the original single-stranded RNA. A
first primer and a second primer are used in the amplification process. A sequence of the first primer or the second primer is sufficiently complementary to a sequence of the specific nucleic acid sequence and a sequence of the first or the second primer is sufficiently homologous to a sequence of the specific nucleic acid sequence. In some instances, both the first primer and second primer are sufficiently complementary and sufficiently homologous to a sequence of the specific nucleic acid sequence. for example, if the specific nucleic acid sequence is double stranded DNA.
The RNA is converted to single-stranded DNA by hybridizing an oligonucleotide primer (the first ~~r 13~0~0~
primer) to the RNA (the first template) and synthesizing a complementary strand of DNA from the first primer, (the first DNA sequence) by using a RNA-directed DNA polymerase. The resulting single-stranded DNA (the second template) is separated from the first template by, for example, hydrolysis of the first template and by using a ribonuclease which is specific for RNA-DNA hybrids (for example, ribonuclease H). The second template is converted to a form which is capable of RNA synthesis by hybridizing a synthetic oligonucleotide (the second primer), which contains at its 3' end a sequence which is sufficiently complementary to the 3' end of the second template and toward its 5' end a sequence containing the antisense strand of a promoter and antisense sequence of a transcription initiation site, and by synthesizing a second DNA sequence covalently attached to the 3' end of the second primer using the second template as a template and synthesizing a third DNA sequence covalently attached to the 3' end of the second template using the second primer as a template, using DNA-directed DNA polymerase. The resulting functional derivative of the second template. which is a third template, is used for the synthesis of a plurality of copies of RNA, the first template, by k ,.,~~
13~n~~7 using a RNA polymerase which is specific for the promoter and transcription initiation site defined by the second primer. Each newly synthesized first template can be converted to further copies of the second template and the third template by repeating the cycle. In addition, repetition of the cycle does not require participation or manipulation by the user.
The amplification process commences with the addition of a suitable template nucleic acid to the appropriate enzymes, primers, and cofactors under the appropriate reaction conditions. This template nucleic acid is in a form which is capable of homogenous and continuous amplification and can function as an intermediate in the cycle set forth in Figure 1. The amplification process involves the net consumption of precursors (primers, ribonucleoside triphosphates and deoxyribonucleoside triphosphates) and the net accumulation of products (RNA and DNA).
The processes of RNA and DNA synthesis will proceed asynchronously until sufficient levels of nucleic acids have been synthesized to allow detection. The amplification process may be monitored by, for example, the synthesis of a labeled product from a labeled precursor.
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It is contemplated that amplification may involve another process either in addition to or in place of the one generally illustrated in Figure 1. Also possible are certain counter-productive enzymatic reactions which occur at permissibly low rates.
Included among the possible non-productive side reactions is the synthesis of RNA and/or DNA in the absence of an added template nucleic acid. Such RNA
and/or DNA products can be discriminated from desired products by determining whether a particular sequence which would be found only between the two priming sites of the specific nucleic acid sequence is present.
The first primer is an oligodeoxyribonucleotide which has at its 3' end a sequence which is sufficiently complementary to the 3' end of the first template. The sequence at the 3' end of the first primer has a particular length and base composition to allow specific and efficient synthesis of the first DNA sequence, under the given conditions of ionic strength and temperature. The first primer may be sufficiently complementary to a region internal to the 3' end of the first template in the fir cycle. In subsequent cycles, the 5' end of the first primer would be complementary to the 3' end of the first d ,.' . .~'C~.v.:~
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template. It is contemplated that the first primer may be composed partially or completely of nucleotides or nucleotide analogs other than the natural deoxyribonucleotides. The 5' end of the first primer may contain sequences which are not complementary to the first template in the first cycle. The non-complementary sequences may be complementary to a nucleic acid which can be immobilized, or to which can be bound a useful non-nucleic acid component, such as a reporter to facilitate detection. Alternatively, the non-complementary sequences may include an antisense sequence of a promoter and an antisense sequence of a transcription initiation site, which could be used for the synthesis of RNA. This RNA
would be complementary to the first template and could be used as an intermediate in another amplification cycle.
The second primer is an oligodeoxyribonucleotide which contains at its 3' end a sequence which is sufficiently complementary to the 3' end of the second template. The second primer has a particular length and base composition to allow specific and efficient synthesis of the second and third DNA sequences, under the given conditions of ionic strength and 1340~i~'l temperature. In addition, the second primer contains the antisense sequence of a functional promoter and the antisense sequence of a transcription initiation site. This sequence, when used as a template for synthesis of the third DNA sequence, contains sufficient information to allow specific and efficient binding of a RNA polymerase and initiation of transcription at the desired site. The promoter sequence may be derived from the antisense strand of a functional promoter. The transcription initiation site may be derived from the 5' terminal sequence of a natural RNA transcript. In the preferred embodiment, the 5'-terminal sequence of the second primer is AATTCTAATACGACTCACTATAGGGAG. This sequence contains the antisense sequence of the promoter and the antisense sequence of the transcription initiation site for T7 RNA polymerase. Alternatively, the transcription initiation site and promoter for another phage RNA polymerase may be used. In addition, sequences which are unrelated to the promoter function may be included at the 5' end of the second primer or between the transcription initiation site and the sequence at the 3' end which hybridizes to the second template. It is contemplated that the second primer may be composed partially or completely of nucleotides or nucleotide analogs other than natural deoxyribonucleotides.
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All of the enzymes used in this invention should meet certain practical specifications. Each enzyme or enzyme preparation should be free of deleterious deoxyribonuclease ("DNase") activities, such as the 5' or 3' exonuclease activities which are often associated with certain DNA polymerases and single-strand or double-strand specific exonuclease or endonucleases. Each enzyme or enzyme preparation should be free of deleterious ribonuclease ("RNase") activities, with the exception of the preferred addition of a ribonuclease activity which is specific for hybrids of RNA and DNA (for example, ribonuclease H). In addition, each enzyme should be reasonably active under the common reaction conditions which are used for the other enzymatic processes, and non-enzymatic processes, such as hybridizing oligonucleotide primers to the RNA or DNA templates.
The DNA-directed RNA polymerase which is used in this invention may be any enzyme capable of binding to a particular DNA sequence called a promoter and specifically initiating in vitro RNA synthesis at a defined initiation site within close proximity to the promoter. The promoter and the initiation site form part of the second primer. In addition the RNA
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polymerase should be capable of synthesizing copies of RNA per functional copy of template in a reasonable amount of time. In the preferred embodiment, the bacteriophage T7 RNA polymerase is used. In addition other bacteriophage RNA
polymerase, such as phage T3, phage III, Salmonella phage sp6, or Pseudomonas phage gh-1 may be used. In another embodiment, other prokaryotic or eukaryotic DNA-directed RNA
polymerase may be used. It should be understood that if alternative RNA polymerases are used, then the necessary changes to the promoter and initiation sequences of the second primer should be made according to the template specificity of the particular RNA polymerase.
The RNA-directed DNA polymerase which is used in this invention may be any enzyme capable of synthesizing DNA from an oligodeoxyribonucleotide primer and a RNA
template. In addition this enzyme may contain activities for DNA-directed DNA polymerase and RNase H. In the preferred embodiment, the avian myeloblastosis viral polymerase ("AMV reverse transcriptase") is used. In addition the RNA-directed DNA polymerase could be from another retrovirus, such as Moloney murine leukemia virus x 13408i~'~
Alternatively, other eukaryotic RNA-directed DNA
polymerases could be used.
The DNA-directed DNA polymerase which is used in this invention may be any enzyme capable of synthesizing DNA from an oligodeoxyribonucleotide primer and a DNA template. This enzyme should not contain either 5'- or 3'- exonuclease activities, which are associated with many types of DNA
polymerase. In the preferred embodiment, the AMV
reverse transcriptase is used. However, other DNA-directed DNA polymerases which naturally lack the 5'- or 3'- exonuclease activities could be used.
These could include certain eukaryotic DNA
polymerases. such as, DNA polymerase ~ or ~ those DNA polymerases which could be isolated from a mammalian tissue, such as calf thymus. An otherwise unsuitable DNA polymerase could be made useful by removing the undesirable exonuclease activities either by alteration of the DNA polymerase gene followed by expression of the altered polymerase in a suitable host cell, or by chemical modification of the DNA
polymerase protein. Altered versions of DNA
polymerase could be made from the Klenow fragment of E. coli DNA polymerase I or the bacteriophage T7 DNA
polymerase. It should be understood that such alternative DNA-directed DNA polymerase activities are added to supplement the activity contributed by the RNA-directed DNA polymerase, since in the preferred embodiment, both RNA-directed and DNA-directed DNA
polymerase activities are supplied by the same enzyme.
The RNase H which could be used in this invention may be any enzyme capable of hydrolyzing a RNA which is annealed to a complementary DNA. This enzyme should not be capable of hydrolyzing single or double-stranded RNA or any DNA. In the preferred embodiment, the E, coli RNase H is used. In addition, - other RNase H enzymes could be used. such as calf thymus RNase H. Since RNase H is an intrinsic activity of AMV reverse transcriptase, the E. coli RNase H will be supplemented in the preferred embodiment by the RNase H of AMV reverse I
I, transcriptase. Alternatively, any other enzyme i capable of separating the second template from the first template could be used.
The above mentioned enzymes and primers are mixed together in a reaction vessel which contains the necessary buffers and cofactors for both DNA and RNA
synthesis. In addition, the ionic conditions and reaction temperature should be compatible with specific hybridization of the primers to the DNA and RNA templates as is known to those skilled in the art. The reaction mixture should be free of such agents which would interfere with the amplification process, specifically substances which could greatly inhibit the activity of the enzymes, interfere with the hybridizing of primers and templates. or degrade non-productively the nucleic acid intermediates and products.
The description of possible detection schemes may be useful to the application of the amplification process. It should be understood that schemes which may be used for detecting the nucleic acids which are synthesized in the amplification process are not limited to those described herein, and it is contemplated that other methods may be used.
In one embodiment, a labeled precursor may be added to the reaction mixture. Amplification is determined by quantitive or qualitative analysis of labeled products. which can be separated from the labeled precursor by using methods known in the art.
,..",. , A labeled nrE?r~mrcnr may ha a ri hnn"~~1 cne; rlc 1 3 4 ~ ~ !~
triphosphate for detecting RNA synthesis, or a deoxynucleoside triphosphate or an oligonucleotide primer for detecting DNA synthesis. The type of label may be a radioisotope or a useful chemical group, such as biotin, a chromophore, a fluorophore, or a hapten which could bind to an antibody, or possibly a protein or an enzyme. The labeled products may be separated from the labeled precursors on the basis of solubility, charge, or size. In addition, the labeled DNA or RNA may be hybridized to a nucleic acid which contains a complementary sequence and which can be immobi lized .
In another embodiment, the products of the amplification process may be bound to an immobilized support, hybridized to a nucleic acid probe containing a complementary sequence. and separated from the unhybridized nucleic acid probe which remains in solution. The products, DNA or RNA, may be bound directly to a solid support by any stable interaction, such as hydrophobic, electrostatic. or covalent interaction. In addition, the products may contain certain chemical groups, for example, biotin, which may be incorporated into the products during the s .,';~::
~~~~~i~~
amplification process to allow binding to an immobilized protein, for example, avidin or streptavidin. In addition, the products may be hybridized to a nucleic acid which contains a complementary sequence and which can be immobilized.
The nucleic acid probe would contain a complementary sequence which forms a sufficiently stable interaction with a product of the amplification process to allow binding under the conditions of hybridization and sustained binding under the conditions used for removal of the unhybridized nucleic acid probe. In the preferred embodiment the complementary sequence would be derived from that part of the specific nucleic acid sequence which is between the sequences of the first primer and the second primer. The nucleic acid probe may be a single-stranded DNA or RNA, or a double-stranded DNA or RNA which can be made single-stranded, or an oligonucleotide which can be composed of deoxyribonucleotides and/or ribonucleotides. In addition, the nucleic acid probe may contain a chemical group which could covalently bind to a product DNA or RNA under the appropriate conditions. The nucleic acid probe may be labeled with a radioisotope or a useful chemical group, such as biotin, a chromophore, a fluorophore, or a hapten ~r~
r:, 'r~.r< .
1340b~'~
which could bind to an antibody. In addition, the nucleic acid probe could be conjugated to a protein or enzyme, for example, a phosphatase or a peroxidase.
In addition, the nucleic acid probe may contain sequences which would allow in vitro replication of the probe.
It is contemplated that the products of the amplification process may be analyzed by methods which are typically used for nucleic acids that have been enriched by molecular cloning techniques. In one alternative, the synthesis of a specific DNA sequence may be detected by digestion of the synthesized DNA
with a restriction endonuclease, followed by electrophoretic separation and detection using methods known in the art. In another alternative, the sequence of amplified RNA may be determined by DNA
synthesis using a RNA-directed DNA polymerase, the first primer, and dideoxynucleoside triphosphates (Stoflet gt al., 1988). In another alternative, the sequence of the amplified third template may be determined by RNA synthesis using the DNA-directed RNA
polymerase used in the amplification process, and 3'-deoxyribonucleoside triphosphates (Axelrod &
Kramer, 1985). In another alternative, the amplified 134080' RNA may encode a polypeptide which could be translated, in vitro. The polypeptide product of the in vitro translation could be analyzed by using an antibody.
A sample suspected of containing or known to contain the specific nucleic acid sequence is added to the reaction mixture in the form of a template nucleic acid which is capable of homogeneous and continuous amplification and may be any intermediate in the cycle set forth in Figure 1. In particular, the template nucleic acid may be a single-stranded RNA which contains at its 5' end a sequence which is sufficiently homologous to that which is at the 3' end of the second primer, and contains a sequence at its 3' end which is sufficiently complementary to the first primer. A template nucleic acid of this form would function as a first template in the amplification process. Alternatively, the template nucleic acid may be a single-stranded DNA which contains at its 3' end a sequence which is sufficiently complementary to at least the 3' end of the second primer, and contains a sequence at its 5' end which is sufficiently homologous to that which is at the 3' end of the first primer. A template nucleic acid of this form would function as a 13408~'r second template in the amplification process.
Alternatively, the template nucleic acid may be a double-stranded DNA, one strand of which contains at its 5' end the entire sequence of the second primer and contains a sequence at its 3' end which is sufficiently complementary to the first primer. The double-stranded DNA functions as a third template in the amplification process.
Although the preparation of a template nucleic acid is not part of the amplification process, the description of possible schemes for generating template nucleic acids may be useful to the application of the amplification process. It should be understood that the schemes which may be used for obtaining the template nucleic acid are not limited to the alternatives which are described herein, and it is contemplated that other methods may be used.
In one alternative, a template nucleic acid which could function as a first template could be a naturally occurring RNA or a RNA fragment which could be generated from a larger RNA molecule by using site specific hydrolysis methods known in the art (Shibahara ~ al., 1987).
1340~~'~
In another alternative, a template nucleic acid which could function as a second template could be generated from a double-stranded DNA by digestion with a restriction endonuclease which has a site immediately flanking the sequence which is sufficiently complementary to the 3' end of the second primer. The resulting double-stranded DNA fragments could then be made single-stranded by using chemical or thermal denaturation methods.
In another alternative, a template nucleic acid which could function as a second template could be generated from a single-stranded DNA or RNA to which has been hybridized an oligonucleotide which is capable of blocking DNA synthesis. This blocking oligonucleotide may contain a chemical group, which could covalently bind to the template, under the appropriate conditions. DNA synthesis from this blocked template using the first primer could result in a synthesized DNA with the same 3' end as the second template. If the original template is RNA, then the resulting DNA-RNA hybrid may be used directly as a template nucleic acid. If the original template is DNA, then the resulting copy of the second template could then be separated from the original template by using chemical or thermal denaturation methods.
. 13~0~~'l In another alternative. a template nucleic acid which could function as a third template could be generated from a single-stranded DNA or RNA by DNA
synthesis from the DNA or RNA template using the second primer. The resulting synthesized DNA could then be separated from the original template by using chemical or thermal denaturation methods. In addition, a RNA template could be hydrolyzed by using chemical or enzymatic methods. The resulting single-stranded DNA has the_sequence of the second primer covalently attached to its 5' end and contains a sequence which is sufficiently complementary to the first primer. This single-stranded DNA could be converted to a transcriptionally functional double-stranded DNA by hybridizing the first primer to the single-stranded DNA, and by synthesizing a DNA
sequence which is covalently attached to the first primer and complementary to the single-stranded DNA.
In a further.alternative, a single-stranded DNA
or RNA template could be obtained from a double-stranded DNA, double-stranded RNA or a DNA-RNA
hybrid by using chemical, thermal, or possibly enzymatic methods. Then, by using one of the alternative schemes proposed above, the resulting iA
l~~Day~~
single-stranded DNA or RNA could then be used to generate a template nucleic acid which could function as a first, second or third template. In addition, an alternative scheme involving the first primer and one strand of nucleic acid, and another alternative scheme involving the second primer and the other (complementary) strand of the nucleic acid may be used concurrently to generate template nucleic acids.
MATERIALS AND METHODS
Materials Oligonucleotides were synthesized using an Applied Biosystems 380A DNA synthesizer. Columns, phosphoramidites, and reagents used for oligonucleotide synthesis were obtained from Applied Biosystems, Inc. through Technical Marketing Associates. Oligonucleotides were purified by polyacrylamide gel electrophoresis followed by DEAF
cellulose chromatography. The radioisotope [d~-32P]
UTP (800 Ci/mmol) was from Amersham. Enzymes for digesting and ligating DNA were purchased from New England Biolabs, and used according to the supplier's 13~OS~=~
recommendations. Preparations containing the large fragment of DNA polymerase 1 (Klenow) were also purchased from New England Biolabs. RNasin and T7 RNA
polymerase from Promega Biotec were purchased through Bio/Can Scientific Inc. Reverse transcriptase and RNase H were obtained from Pharmacia. The supplier for proteinase K was Boehringer Mannheim Canada. E.
coli strain HB101 (ATCC 33694) was used for all transformations. The plasmid pUCl9 (Norrander ~ al., 1983) was purchased from Bethesda Research Laboratories.
Isolation of DNA and sequencing E. coli transformants were grown on YT medium (Miller, 1972) containing 50 ug/ml ampicillin.
Plasmid DNA was purified by a rapid boiling method (Holmes and Quigley, 1981). DNA fragments and vectors used for all constructions were separated by electrophoresis on low melting point agarose, and purified from the molten agarose by phenol extraction and ethanol precipitation (Maniatis g~ al., 1982).
Plasmid DNA was sequenced using a modification (Hattori ~ ~. 1985) of the dideoxy method (Sanger g~ al., 1977). Reactions were run using the -20 universal primer (New England Biolabs).
1~~OSi~~
TCA precipitation Aliquots (5u1) of amplification reactions were quenched in 20u1 10 mM EDTA and placed on ice until all time point samples had been collected. The quenched samples were then applied to glass filter discs, and immediately dropped into ice-cold 5%
trichloroacetic acid ("TCA") - 1% sodium pyrophosphate for 10 min with occasional mixing. Two 5 min washes with ice-cold 5% TCA were followed by two additional washes with 95% ethanol and lyophilization to dryness. Radioactivity was determined in a liquid scintillation counter.
Polyacrylamide gel electrophoresis Samples (1 to 6 ul) were mixed with 4-5 ul formamide dye (90% deionized formamide, 10 mM TrisHCl (pH 8.0), 1 mM EDTA, xylene cyanol and bromophenol blue), and applied to a pre-run 12-cm- long 7%
denaturing polyacrylamide gel. Gels were run at 350 volts until the bromophenol blue dye had reached the bottom. In some cases the gels were fixed and dried prior to autoradiography. Fixing involved a 15 min wash in 10% methanol- 7% acetic acid. The profiles of ~~4~~~' the RNA products separated by this procedure were visualized by autoradiography at room temperature.
Example 1: Design and synthesis of oligonucleotides for a gag test system A synthetic DNA sequence (Figure 2A) was designed to include an EcoRI site, a T7 phage promoter, a sequence required for initiation of transcription by T7 RNA polymerase and a 19 by hybridization region (hybridization region 1). The 47 b antisense strand oligonucleotide (T7Hl.GAG) involved in the cloning of these elements also serves as the first primer.
Hybridization region 2 lies 53 by away from hybridization region 1 and is 20 by in length. The primer made to this region (H2.GAG) is a 20 b oligonucleotide duplicate of the sense strand and is not used for cloning. The sequence spanning and including the hybridization regions is a 92 by segment of the gag portion of the HIV-1 genome. This particular gene segment was chosen because the primers were predicted to hybridize efficiently and because the distance between the two hybridization regions was relatively short. In addition, a XbaI site was placed at the end of the sequence for cloning ease. The gag ~3~08~~
test sequence also contains SphI and PstI sites which can aid in the screening of recombinants.
A total of four oligonucleotides were used in the cloning of this fragment. N1.GAG, used in the construction of both the gag test and the gag2 test sequence, completes the antisense strand and is used only in the cloning process. Similarly, T74.PR0 is the sense strand component of the T7 promoter. N2.GAG
however, was used in the construction of both test fragments, and has also been used as an intermediate (second template) in two steps of the amplification cycle. The entire cloned gag test fragment can also represent an intermediate of the amplification cycle (third template). Once cloned into an appropriate vector the gag test DNA could be transcribed by T7 RNA
polymerase to produce a RNA fragment (first template) useful as an amplification intermediate involved in three of the steps. In addition, T7H1.GAG and H2.GAG
serve as primers in the test system.
The gag2 test synthetic DNA fragment (Figure 2B) does not contain the T7 promoter, but the remainder of the sequence is identical to the gag test sequence and therefore, both N1.GAG and N2.GAG were involved in its x ;~
'~ v..
134U8~' construction. The oligonucleotide required to complete the antisense strand is called H1.GAG. Once cloned, the gag2 test fragment can be used as a template for testing amplification, using a DNA
restriction fragment as template nucleic acid.
Example 2: Construction of the gag test plasmids The oligonucleotides T74.PR0 and N1.GAG (2 ug each), were phosphorylated separately in 20 ul reactions containing 70 mM TrisHCl (pH 7.6), 10 mM
MgCl2, 5 mM DTT, 0.5 mM ATP and 5 units T4 polynucleotide kinase, at 37°C for 30 min.
Phosphorylated T74.PR0 and N1.GAG (10 ul of each) were mixed with 1 ug each of unphosphorylated T7Hl.GAG and N2.GAG, and 3 ul 100 mM TrisHCl (pH7.8) - 500 mM NaCl, in a final volume of 29 ul for the gag test assembly.
The gag2 test mixture contained 10 ul phosphorylated N1.GAG, 1 ug each of unphosphorylated H1.GAG and N2.GAG, and 1.8 ul 100 mM TrisHCl (pH 7.8) - 500 mM
NaCl, in a final volume of 18 ul. The oligonucleotide mixtures were hybridized separately by placing them at 90°C for 10 min followed by slow cooling to room temperature for 10 to 16 h 60 ul reactions containing 50 mM TrisHCl (pH 7.8), 10 mM MgCl2, 20 mM DTT, 1mM
13~0~~'I
ATP and 50 ug/ml BSA were used to ligate the hybridized oligonucleotides together. 400 units T4 DNA ligase was added to the gag test reaction and it was incubated at 15°C for 2h while the gag2 test reaction was incubated for 14 to 16 h with 200 units T4 DNA ligase.
The isolated and purified synthetic DNA segments were mixed with plasmid pUCl9 which had been linearized by digestion at restriction enzyme sites within the polylinker region. T4 DNA ligase was used to ligate the gag test sequence into the EcoRI- XbaI
fragment of pUCl9, while the gag2 test sequence was ligated to the SmaI-XbaI fragment. Plasmid DNA from transformants obtained after these reactions were used to transform E. coli were screened by restriction analysis , and the final plasmids (pGAG.TEST and pGAG2.TEST) were determined to be correct by sequence analysis.
Example 3: Effect of primer concentration on RNA
amplification The reaction mixtures (25 ul) which were used to amplify RNA transcribed from the gag test ,..Y I
1340~0'r oligonucleotides contained 50 nM TrisHC1 (pH
8.45), 6 mM MgCl2 40 mM KC1, 10 mM
dithiothreitol, 0.5 mM NTP (ATP, CTP, GTP, UTP), 1 mM dNTP (dATP, dCTP, dGTP, dTTP), 20 units RNasin, 10 units T7 RNA polymerase, 10 units reverse transcriptase, 0.4 units RNase H, and 10 uCi [ a -32P] UTP. Two of the reactions contained 0.5 ng (0.015 pmoles) N2.GAG while the other two reactions contained no template. The primers T7Hl.GAG and H2.GAG were each added at final concentrations of 3.4 uM or 0.34 uM to reactions containing either N2.GAG or no template. The reactions were incubated at 42°C
for 2h. Total synthesis of RNA was monitored by determining the incorporation of TCA insoluble cpm at 30 min. intervals. The effect of the primer concentration on template-dependent RNA
synthesis is shown in Table 1. Aliquots of each reaction, containing equal amounts of synthesized RNA, were analyzed by PAGE and autoradiography (Figure 3, lanes 1-4 numbered the same as the reactions).
Table 1: RNA amplification after 2 h.
Concentration of Template RNA
Reaction each primier (ng) Synthesized (uM) (ug) 1 3.4 0.5 2.8 2 3.4 - 2.1 3 0.34 0.5 1.8 4 0.34 - 0,-7 13408i~'7 It was found that while reaction 1 resulted in the greatest incorporation of isotope, the no template control, reaction 2, was also high (73% of reaction 1) and produced a very similar electrophoretic profile.
It would therefore appear that in the presence of high primer concentrations, a RNA transcript of identical size to that expected in amplification is produced in the absence of any template. Results using samples with a 10- fold decrease in primer concentration were dramatically different. The amount of RNA produced in reaction 3 was 2.6 times that of reaction 4, but while virtually all of the transcript was found in a single band of the expected size in reaction 3, no fragments greater than 60 to 70 b were found in reaction 4.
Primer concentration therefore plays a significant role in the accuracy and efficiency of RNA
amplification.
A control RNA transcript used to represent the size of fragment expected to be generated by the amplification system (lane 0 of Figure 3) was prepared by transcription from the test plasmid. pGAG.TEST was linearized by digestion with XbaI, proteinase K
treated (Maniatis g~ al., 1982), phenol extracted and ethanol precipitated. T7 RNA polymerase was then used 134~8~'~
according to the supplier's recommendations to transcribe 0.5 ug of the resulting fragment in a 25 ul reaction mixture containing 10 uCi [ a' -32P] UTP.
example 4: Effect of template concentration on RNA
amplification The standard 50 ul reaction mixture used to amplify RNA transcribed from the gag test oligonucleotides contained 0.34 uM T7H1.GAG, 0.34 uM
H2.GAG, 50 mM TrisHCl (pH 8.45), 6 mM MgCl2, 40 mM
KC1, 10 mM DTT, 0.5 mM NTP, 1 mM dNTP, 40 units RNasin, 20 units T7 RNA polymerase, 20 units reverse transcriptase, 0.8 units RNase H and 10 - 20 uCi [ ~-32P] UTP. The reactions contained amounts of template (N2.GAG) varying from 1 ng to 1 fg. One reaction contained no template. The reactions were incubated at 42°C for 3h, during which total synthesis of RNA was monitored by determining the incorporation of TCA insoluble cpm at 30 min intervals. As indicated in Table 2, total RNA synthesis was higher than the no template control for all template concentrations tested. Although the total synthesis of RNA generally decreased with decreasing template concentration, this decrease in synthesis was not Z~40~0'l quantitative. Thus, the degree of amplification of RNA per starting template generally increased with decreasing template concentration. Amplification of 8 x 108 fold was achieved by synthesis of 0.8 ug RNA
from 1 fg of N2.GAG template. One fg of the 102 b N2.GAG oligonucleotide represents approximately 2 a 104 molecules.
Table 2: RNA amplification from N2.GAG after 3h.
RNA
Synthesized Fold Reaction Template (ug) amplifi cation 1 1 ng 3.5 3.5 x 103 2 100 pg 4.4 4.4 x 104 3 10 pg 4.1 4.1 x 105 4 1 pg 3.0 3.0 x 106 5 100 fg 2.7 2.7 x 107 6 10 fg 1.9 1.9 x 108 7 1 fg 0.78 7.8 x 108 8 - 0Ø46 -The RNA which was synthesized after a reaction time of 3h was analyzed by PAGE, for each template concentration (Figure 4 lanes 1-8, numbered the same as the reactions). A major band representing a RNA of about 100 b was present in all reactions except the ones containing 1 fg template and no template. The reaction containing 1 fg template did not have much of this 100 b product at 3 h but the overall RNA
.1~~C~~i~'l synthesis was higher than and qualitatively different from the no template reaction.
Example 5: Hybridization analysis of RNA products.
Amplification reactions containing amounts of N2.GAG template varying from 1 pg to 0.1 fg were performed following the teaching of example 4, except the radiolabeled UTP was omitted. The reactions were incubated at 42°C for 3 h. Aliquots were removed from each reaction at 30 min intervals and applied to a nylon membrane (Amersham). The nucleic acids that were contained in these reaction aliquots were fixed by exposure to ultraviolet light. The membrane was prehybridized at 50°C for 1 h in prehybridization buffer consisting of a final concentration of 50% v/v formamide, 5 X SSC and 5 X Denhardt's solution (Maniatis ~ ~. 1982; Southern g~ al., 1975) at a volume equivalent to 5 mls of solution per 100 cm2 and hybridized with a radiolabeled probe with a specific activity of 106 cpm/ml of hybridization solution. Hybridization was performed at 50°C for 16 h in 50% formamide, 5 X SSC and 5 X Denhardt's solution (Maniatis g~ ~. 1982; Southern g~ al., 1975). The radiolabeled probe was the synthetic ~3408J7 oligonucleotide 5' GATCTGGGATAGAGTACATCCA 3' which had been labeled at the 5' end using T4 polynucleotide kinase and ( Y - 3zP) ATP. After the membrane was washed at 50°C in a series of 2, 3 min. washes consisting of 2 X SSC, 0.1$ v/v SDS and 0.2 X SCC, 0.1~ v/v SDS (Southern et al., 1975; Maniatis et al., 1982; Szostak et al., 1979).
Figure 5 shows the results of the hybridization analysis performed on the amplification reactions, containing various amounts of N2.GAG template, which were sampled at different times of incubation.
Each column of Figure 5 represents a different time point (l, 30 min; 2, 60 min; 3, 90 min; 4, 120 min; 5, 150 min; 6, 180 min) and each row represents a different amount of added N2.GAG template (1, lpg; 2, 100 fg; 3, 10 fg; 4, 1 fg; 5, 0.1 fg; 6, no template). Amplification of nucleic acids which hybridized to the labeled probe were observed for rows 1-3 (1 pg - 10 fg), however the hybridization to specific nucleic acids in rows 4 - 5 (1 fg, 0.1 fg) was not higher than row 6 (no template). The apparent non-specific binding of labeled probe in row 6 seems to be related to DNA or RNA synthesis since the hybridization signal increases with increasing time.
.-13~U8~~
Example 6: Use of DNA restriction fragment as template.
The plasmid pGAG2.TEST was digested with MspI and proteinase K, purified by phenol extraction and ethanol precipitation, and denatured by boiling for 5 min. Amplification reactions were performed and analyzed following the teaching of Example 4, except the MspI digested pGAG2.TEST was used as a template instead of the N2.GAG oligonucleotide. The amounts of plasmid added to each reaction varied from 55 ng to 5.5 pg, and no template. To simulate additional DNA
which would be present in an actual sample, alternate reactions contained 1 ng of calf thymus DNA which had been similarly digested, purified and denatured.
After a 3 h incubation at 42°C, the synthesis of RNA
was determined by TCA precipitation and PAGE
analysis. As indicated in Table 3, total RNA
synthesis was higher than the no template controls for all template concentrations tested. The degree of amplification was calculated based on RNA synthesis from the actual template which was 1.8% of the total plasmid DNA.
The total RNA synthesis (degree of amplification) from a particular initial level template concentration ~r~, m:,, 1340~0'~
was consistently lower for the restriction.fragment (Table 3) as compared to that for the synthetic oligonucleotide template (Table 2). This could be due to competition with the complementary strand of the restriction fragment template under the conditions used.
Table 3: RNA amplification from MspI-digested pGAG2.TEST
RNA Fold Reaction Template* Synth esized** amplific ation**
1 55.0 ng [ 1 ng] 3.65 3.7 x 103 2 (4.05) (4.1 x 103) 3 5.5 ng [100 pg] 3.54 3.5 x 104 4 (3.16) (3.2 x 104) 5 550.0 pg [ 10 pg] 2.29 2.3 x 105 6 (2.79) (2.8 x 105) 7 55.0 pg [ 1 pg] 2.62 2.6 x 106 8 (0.67) (0.7 x 106) 9 5.5 pg [100 fg] 1.37 1.4 x 107 10 (2.26) (2.3 x 107) 11 - 1.25 -12 (0.08) 2 0 * Numbersis brackets indicate valent amountsof GAG.
equi N2.
** Numbers in parentheses indicateRNA synthesisin ce of presen 1 ug MspI-di gested calf thymus DNA.
,. ~ 1340~i~'~
The RNA which was synthesized after a reaction time of 3h was analyzed by PAGE (Figure 6, lanes 1-6, 11 and 12, numbered the same as the reactions). The major band representing a RNA of about 100 b was present in reactions (lanes) 1-6 but absent in the no template reactions (lanes 11 and 12). The RNA in lane 0 was a standard, which was prepared following the teaching of Example 3. There was no apparent qualitative difference in the synthesized RNA either with (lanes 2, 4 and 6) or without (lanes 1, 3, and 5) the additional of 1 ~g of MspI-digested calf thymus DNA.
Although preferred embodiments of the invention have been described in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from either the spirit of the invention or the scope of the appended claims.
~.. 1340~i~r~
References 1. Abbott, M.A., Poiesz, B.J., Byrne, B.C., Kwok, S., Sninsky, J.J., and Ehrlich, G.D. (1988), J. Inf.
Diseases ~$, 1158 2. Axelrod, V.D. and Kramer, F.R. (1985) Biochemistry 24, 5716-5723.
3. Biebricher, C.K. (1987) Cold Spring Harbor Symp.
Quant. Biol. 52, 299-306 4. Bugawan, T.L., Saiki, R.K., Levenson, C.H., Watson, R.M. and Erlich, H.A. (1988) Biotechnology _6, 943-947 5. Chu, B.C.F., Kramer, F.R. and Orgel, L.E. (1986) Nucleic Acids Res. 14, 5591-5603 6. Denhardt, D.T. (1966) Biochem. Biophy. Res. Commun.
_2~ 641-646 7. Harbarth, P. and Vosberg, H.P. (1988) DNA 7, 297-306 8. Hattori, M., Hidaka, S., and Sakaki, Y. (1985) Nucleic Acids Res. 13, 7813-7827.
9. Holmes, D.S. and Quigley, M. (1981) Anal. Biochem.
114, 193-197.
10. Kawaski, E.S., Clark, S.S., Coyne, M.Y., Smith, S.D., Champlin, R., Witte, O.N. and McCormick, F.P. (1988) Proc. Nat'1. Acad. Sc. U.S.A. ~1_, 5698-5702 11. Klenow, H. and I. Henningsen (1970) Proc. Nat'1. Acad.
Sc. U.S.A. 65, 168 12. Lizardi, R.M., Guerra, C.E., Lomeli, H., Tussie-Luna, I, and Kramer, F.R. (1988) Biotechnology f, 1197-1202 13. Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
SUN~1ARY OF THE INVENTION
This invention relates to an amplification process which is expedient and requires less participation and fewer manipulations by the user of the process than conventional amplification processes. The amplification takes place at a relatively constant ambient temperature. In addition, each cycle of the process generates a plurality of copies of product from one substrate. The amplification process of this invention may be used to increase the quantity of a specific nucleic acid thus circumventing the copy number problem. Hence, probe assays may be more readily used. The amplification process could also be used to increase the purity of a specific nucleic acid sequence as a substitute for conventional cloning methodology.
One aspect of the invention is a process for amplifying a specific nucleic acid sequence. The process involves the synthesis of single-stranded RNA, single-stranded DNA, and double stranded DNA. The single stranded RNA is a first template for a first primer. The single stranded DNA is a second template for a second ~3~O~u7 primer. The double stranded DNA is a third template for the synthesis of a plurality of copies of the first template. A sequence of the first or the second primer is sufficiently complementary to a sequence of the specific nucleic acid sequence and a sequence of the first or the second primer is sufficiently homologous to a sequence of the specific nucleic acid sequence. A 3' end of the first primer is oriented towards a 3' end of the second primer on complementary strands.
Another aspect of the invention is a process for amplifying a specific nucleic acid sequence. The process involves:
(a) hybridizing a first primer to a first template. The first primer has a DNA
sequence which is sufficiently complementary to a RNA sequence of the first template;
(b) synthesizing a first DNA sequence covalently attached to the first primer and complementary to the RNA sequence of the first template. The first DNA sequence and the first primer comprise a second template;
_ g _ (c) separating the first template from the second template to allow hybridization of a second primer;
(d) hybridizing the second primer to the second template. The second primer has a DNA
sequence which is sufficiently complementary to a DNA sequence of the second template.
(e) synthesizing a second DNA sequence covalently attached to the second primer and complementary to the DNA sequence of the second template and synthesizing a third DNA
sequence covalently attached to the second template and complementary to the DNA
sequence of the second primer. The second and third DNA sequences, the second primer and the second template comprise a third template;
_ g _ 13~U~~~
- to -(f~ synthesizing a plurality of copies of the RNA sequence of the first template from the third template.
A sequence of the first or the second primer is sufficiently complementary to a sequence of the specific nucleic acid sequence and a sequence of the first or the second primer is sufficiently homologous to a sequence of the specific nucleic acid sequence.
A 3' end of the first primer is oriented towards a 3' end of the second primer on complementary strands.
In a further alternative of the invention, the second primer of DNA has a sequence at its 3' end which is sufficiently complementary to the DNA
sequence of the second template. The second primer has at its 5' end an antisense sequence of a promoter and an antisense sequence of a transcription initiation site for a RNA polymerase.
13~08i37 Another alternative of the invention is a process for amplifying a specific nucleic acid sequence. The process involves combining a first primer, a second primer, ribonuclease H, a RNA-directed DNA polymerase, a DNA-directed DNA polymerase, a RNA polymerase, ribonucleoside triphosphates and deosyribonucleoside triphosphates with a sample. The first primer of DNA
has a sequence which is sufficiently complementary to a first template of RNA. The second primer of DNA has a sequence which is sufficiently complementary to a second template of DNA, and an antisense sequence of a promoter and an antisense sequence of a transcription initiation site which are recognized as substrate by the RNA polymerase. A sequence of the first primer or the second primer is sufficiently complementary to a sequence of the specific nucleic acid sequence and a sequence of the first primer or the second primer is sufficiently homologous to a sequence of the specific nucleic acid. A 3' end of the first primer is oriented towards a 3' end of the second primer on complementary strands.
A further alternative of the invention is a process for amplifying a specific nucleic acid sequence. The process involves adding a first .,.....
primer, a second primer, avian myeloblastosis viral polymerase, E. coli ribonuclease H, bacteriophage T7 RNA polymerase, ribonucleoside triphosphates and deoxyribonucleoside triphosphates to a sample. The first primer of DNA has a sequence which is sufficiently complementary to a first template of RNA. The second primer of DNA has a sequence which is sufficiently complementary to a second template of DNA, and an antisense sequence of a promoter and an antisense sequence of a transcription initiation site which are recognized as substrate by T7 RNA polymerase. A sequence of the first primer or the second primer is sufficiently complementary to a sequence of the specific nucleic acid sequence and a sequence of the first primer or the second primer is sufficiently homologous to a sequence of the specific nucleic acid sequence. A 3' end of the first primer is oriented towards a 3' end of the second primer on complementary strands.
BRIEF DESCRIPTION OF THE DRAWINGS
In drawings which illustrate embodiments of the invention, x 134080' Figure 1 is a general illustration of the nucleic acid amplification process;
Figure 2 shows the synthetic oligonucleotide DNA
sequences which are used for testing the amplification process: Figure 2A, the gag test sequence; Figure 2B, the gag2 test sequence;
Figure 3 is an autoradiogram of PAGE analysis of amplification reactions using different primer concentrations;
Figure 4 is an autoradiogram of PAGE analysis of amplification reactions using different template concentrations;
Figure 5 is an autoradiogram of Dot-blot hybridization on amplification reactions;
Figure 6 is an autoradiogram of PAGE analysis of amplification reaction using restriction fragments as template;
1340~~'7 Figure 7 is a general illustration of the indirect nucleic acid amplification process.
~.,~°~~,i~;
.»
1340~~'~
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
This invention relates to a process for amplifying a specific nucleic acid sequence. The amplification involves the alternate synthesis of DNA
and RNA and is generally illustrated in Figure 1. In this process. single-stranded RNA is converted to single-stranded DNA which in turn is converted to a functional template for the synthesis of a plurality of copies of the original single-stranded RNA. A
first primer and a second primer are used in the amplification process. A sequence of the first primer or the second primer is sufficiently complementary to a sequence of the specific nucleic acid sequence and a sequence of the first or the second primer is sufficiently homologous to a sequence of the specific nucleic acid sequence. In some instances, both the first primer and second primer are sufficiently complementary and sufficiently homologous to a sequence of the specific nucleic acid sequence. for example, if the specific nucleic acid sequence is double stranded DNA.
The RNA is converted to single-stranded DNA by hybridizing an oligonucleotide primer (the first ~~r 13~0~0~
primer) to the RNA (the first template) and synthesizing a complementary strand of DNA from the first primer, (the first DNA sequence) by using a RNA-directed DNA polymerase. The resulting single-stranded DNA (the second template) is separated from the first template by, for example, hydrolysis of the first template and by using a ribonuclease which is specific for RNA-DNA hybrids (for example, ribonuclease H). The second template is converted to a form which is capable of RNA synthesis by hybridizing a synthetic oligonucleotide (the second primer), which contains at its 3' end a sequence which is sufficiently complementary to the 3' end of the second template and toward its 5' end a sequence containing the antisense strand of a promoter and antisense sequence of a transcription initiation site, and by synthesizing a second DNA sequence covalently attached to the 3' end of the second primer using the second template as a template and synthesizing a third DNA sequence covalently attached to the 3' end of the second template using the second primer as a template, using DNA-directed DNA polymerase. The resulting functional derivative of the second template. which is a third template, is used for the synthesis of a plurality of copies of RNA, the first template, by k ,.,~~
13~n~~7 using a RNA polymerase which is specific for the promoter and transcription initiation site defined by the second primer. Each newly synthesized first template can be converted to further copies of the second template and the third template by repeating the cycle. In addition, repetition of the cycle does not require participation or manipulation by the user.
The amplification process commences with the addition of a suitable template nucleic acid to the appropriate enzymes, primers, and cofactors under the appropriate reaction conditions. This template nucleic acid is in a form which is capable of homogenous and continuous amplification and can function as an intermediate in the cycle set forth in Figure 1. The amplification process involves the net consumption of precursors (primers, ribonucleoside triphosphates and deoxyribonucleoside triphosphates) and the net accumulation of products (RNA and DNA).
The processes of RNA and DNA synthesis will proceed asynchronously until sufficient levels of nucleic acids have been synthesized to allow detection. The amplification process may be monitored by, for example, the synthesis of a labeled product from a labeled precursor.
A
It is contemplated that amplification may involve another process either in addition to or in place of the one generally illustrated in Figure 1. Also possible are certain counter-productive enzymatic reactions which occur at permissibly low rates.
Included among the possible non-productive side reactions is the synthesis of RNA and/or DNA in the absence of an added template nucleic acid. Such RNA
and/or DNA products can be discriminated from desired products by determining whether a particular sequence which would be found only between the two priming sites of the specific nucleic acid sequence is present.
The first primer is an oligodeoxyribonucleotide which has at its 3' end a sequence which is sufficiently complementary to the 3' end of the first template. The sequence at the 3' end of the first primer has a particular length and base composition to allow specific and efficient synthesis of the first DNA sequence, under the given conditions of ionic strength and temperature. The first primer may be sufficiently complementary to a region internal to the 3' end of the first template in the fir cycle. In subsequent cycles, the 5' end of the first primer would be complementary to the 3' end of the first d ,.' . .~'C~.v.:~
13400?
template. It is contemplated that the first primer may be composed partially or completely of nucleotides or nucleotide analogs other than the natural deoxyribonucleotides. The 5' end of the first primer may contain sequences which are not complementary to the first template in the first cycle. The non-complementary sequences may be complementary to a nucleic acid which can be immobilized, or to which can be bound a useful non-nucleic acid component, such as a reporter to facilitate detection. Alternatively, the non-complementary sequences may include an antisense sequence of a promoter and an antisense sequence of a transcription initiation site, which could be used for the synthesis of RNA. This RNA
would be complementary to the first template and could be used as an intermediate in another amplification cycle.
The second primer is an oligodeoxyribonucleotide which contains at its 3' end a sequence which is sufficiently complementary to the 3' end of the second template. The second primer has a particular length and base composition to allow specific and efficient synthesis of the second and third DNA sequences, under the given conditions of ionic strength and 1340~i~'l temperature. In addition, the second primer contains the antisense sequence of a functional promoter and the antisense sequence of a transcription initiation site. This sequence, when used as a template for synthesis of the third DNA sequence, contains sufficient information to allow specific and efficient binding of a RNA polymerase and initiation of transcription at the desired site. The promoter sequence may be derived from the antisense strand of a functional promoter. The transcription initiation site may be derived from the 5' terminal sequence of a natural RNA transcript. In the preferred embodiment, the 5'-terminal sequence of the second primer is AATTCTAATACGACTCACTATAGGGAG. This sequence contains the antisense sequence of the promoter and the antisense sequence of the transcription initiation site for T7 RNA polymerase. Alternatively, the transcription initiation site and promoter for another phage RNA polymerase may be used. In addition, sequences which are unrelated to the promoter function may be included at the 5' end of the second primer or between the transcription initiation site and the sequence at the 3' end which hybridizes to the second template. It is contemplated that the second primer may be composed partially or completely of nucleotides or nucleotide analogs other than natural deoxyribonucleotides.
a ,~j l~~d 1~~O~i~'~
All of the enzymes used in this invention should meet certain practical specifications. Each enzyme or enzyme preparation should be free of deleterious deoxyribonuclease ("DNase") activities, such as the 5' or 3' exonuclease activities which are often associated with certain DNA polymerases and single-strand or double-strand specific exonuclease or endonucleases. Each enzyme or enzyme preparation should be free of deleterious ribonuclease ("RNase") activities, with the exception of the preferred addition of a ribonuclease activity which is specific for hybrids of RNA and DNA (for example, ribonuclease H). In addition, each enzyme should be reasonably active under the common reaction conditions which are used for the other enzymatic processes, and non-enzymatic processes, such as hybridizing oligonucleotide primers to the RNA or DNA templates.
The DNA-directed RNA polymerase which is used in this invention may be any enzyme capable of binding to a particular DNA sequence called a promoter and specifically initiating in vitro RNA synthesis at a defined initiation site within close proximity to the promoter. The promoter and the initiation site form part of the second primer. In addition the RNA
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~k.",a~~~ls.~
isY.r..
1340~i~'~
polymerase should be capable of synthesizing copies of RNA per functional copy of template in a reasonable amount of time. In the preferred embodiment, the bacteriophage T7 RNA polymerase is used. In addition other bacteriophage RNA
polymerase, such as phage T3, phage III, Salmonella phage sp6, or Pseudomonas phage gh-1 may be used. In another embodiment, other prokaryotic or eukaryotic DNA-directed RNA
polymerase may be used. It should be understood that if alternative RNA polymerases are used, then the necessary changes to the promoter and initiation sequences of the second primer should be made according to the template specificity of the particular RNA polymerase.
The RNA-directed DNA polymerase which is used in this invention may be any enzyme capable of synthesizing DNA from an oligodeoxyribonucleotide primer and a RNA
template. In addition this enzyme may contain activities for DNA-directed DNA polymerase and RNase H. In the preferred embodiment, the avian myeloblastosis viral polymerase ("AMV reverse transcriptase") is used. In addition the RNA-directed DNA polymerase could be from another retrovirus, such as Moloney murine leukemia virus x 13408i~'~
Alternatively, other eukaryotic RNA-directed DNA
polymerases could be used.
The DNA-directed DNA polymerase which is used in this invention may be any enzyme capable of synthesizing DNA from an oligodeoxyribonucleotide primer and a DNA template. This enzyme should not contain either 5'- or 3'- exonuclease activities, which are associated with many types of DNA
polymerase. In the preferred embodiment, the AMV
reverse transcriptase is used. However, other DNA-directed DNA polymerases which naturally lack the 5'- or 3'- exonuclease activities could be used.
These could include certain eukaryotic DNA
polymerases. such as, DNA polymerase ~ or ~ those DNA polymerases which could be isolated from a mammalian tissue, such as calf thymus. An otherwise unsuitable DNA polymerase could be made useful by removing the undesirable exonuclease activities either by alteration of the DNA polymerase gene followed by expression of the altered polymerase in a suitable host cell, or by chemical modification of the DNA
polymerase protein. Altered versions of DNA
polymerase could be made from the Klenow fragment of E. coli DNA polymerase I or the bacteriophage T7 DNA
polymerase. It should be understood that such alternative DNA-directed DNA polymerase activities are added to supplement the activity contributed by the RNA-directed DNA polymerase, since in the preferred embodiment, both RNA-directed and DNA-directed DNA
polymerase activities are supplied by the same enzyme.
The RNase H which could be used in this invention may be any enzyme capable of hydrolyzing a RNA which is annealed to a complementary DNA. This enzyme should not be capable of hydrolyzing single or double-stranded RNA or any DNA. In the preferred embodiment, the E, coli RNase H is used. In addition, - other RNase H enzymes could be used. such as calf thymus RNase H. Since RNase H is an intrinsic activity of AMV reverse transcriptase, the E. coli RNase H will be supplemented in the preferred embodiment by the RNase H of AMV reverse I
I, transcriptase. Alternatively, any other enzyme i capable of separating the second template from the first template could be used.
The above mentioned enzymes and primers are mixed together in a reaction vessel which contains the necessary buffers and cofactors for both DNA and RNA
synthesis. In addition, the ionic conditions and reaction temperature should be compatible with specific hybridization of the primers to the DNA and RNA templates as is known to those skilled in the art. The reaction mixture should be free of such agents which would interfere with the amplification process, specifically substances which could greatly inhibit the activity of the enzymes, interfere with the hybridizing of primers and templates. or degrade non-productively the nucleic acid intermediates and products.
The description of possible detection schemes may be useful to the application of the amplification process. It should be understood that schemes which may be used for detecting the nucleic acids which are synthesized in the amplification process are not limited to those described herein, and it is contemplated that other methods may be used.
In one embodiment, a labeled precursor may be added to the reaction mixture. Amplification is determined by quantitive or qualitative analysis of labeled products. which can be separated from the labeled precursor by using methods known in the art.
,..",. , A labeled nrE?r~mrcnr may ha a ri hnn"~~1 cne; rlc 1 3 4 ~ ~ !~
triphosphate for detecting RNA synthesis, or a deoxynucleoside triphosphate or an oligonucleotide primer for detecting DNA synthesis. The type of label may be a radioisotope or a useful chemical group, such as biotin, a chromophore, a fluorophore, or a hapten which could bind to an antibody, or possibly a protein or an enzyme. The labeled products may be separated from the labeled precursors on the basis of solubility, charge, or size. In addition, the labeled DNA or RNA may be hybridized to a nucleic acid which contains a complementary sequence and which can be immobi lized .
In another embodiment, the products of the amplification process may be bound to an immobilized support, hybridized to a nucleic acid probe containing a complementary sequence. and separated from the unhybridized nucleic acid probe which remains in solution. The products, DNA or RNA, may be bound directly to a solid support by any stable interaction, such as hydrophobic, electrostatic. or covalent interaction. In addition, the products may contain certain chemical groups, for example, biotin, which may be incorporated into the products during the s .,';~::
~~~~~i~~
amplification process to allow binding to an immobilized protein, for example, avidin or streptavidin. In addition, the products may be hybridized to a nucleic acid which contains a complementary sequence and which can be immobilized.
The nucleic acid probe would contain a complementary sequence which forms a sufficiently stable interaction with a product of the amplification process to allow binding under the conditions of hybridization and sustained binding under the conditions used for removal of the unhybridized nucleic acid probe. In the preferred embodiment the complementary sequence would be derived from that part of the specific nucleic acid sequence which is between the sequences of the first primer and the second primer. The nucleic acid probe may be a single-stranded DNA or RNA, or a double-stranded DNA or RNA which can be made single-stranded, or an oligonucleotide which can be composed of deoxyribonucleotides and/or ribonucleotides. In addition, the nucleic acid probe may contain a chemical group which could covalently bind to a product DNA or RNA under the appropriate conditions. The nucleic acid probe may be labeled with a radioisotope or a useful chemical group, such as biotin, a chromophore, a fluorophore, or a hapten ~r~
r:, 'r~.r< .
1340b~'~
which could bind to an antibody. In addition, the nucleic acid probe could be conjugated to a protein or enzyme, for example, a phosphatase or a peroxidase.
In addition, the nucleic acid probe may contain sequences which would allow in vitro replication of the probe.
It is contemplated that the products of the amplification process may be analyzed by methods which are typically used for nucleic acids that have been enriched by molecular cloning techniques. In one alternative, the synthesis of a specific DNA sequence may be detected by digestion of the synthesized DNA
with a restriction endonuclease, followed by electrophoretic separation and detection using methods known in the art. In another alternative, the sequence of amplified RNA may be determined by DNA
synthesis using a RNA-directed DNA polymerase, the first primer, and dideoxynucleoside triphosphates (Stoflet gt al., 1988). In another alternative, the sequence of the amplified third template may be determined by RNA synthesis using the DNA-directed RNA
polymerase used in the amplification process, and 3'-deoxyribonucleoside triphosphates (Axelrod &
Kramer, 1985). In another alternative, the amplified 134080' RNA may encode a polypeptide which could be translated, in vitro. The polypeptide product of the in vitro translation could be analyzed by using an antibody.
A sample suspected of containing or known to contain the specific nucleic acid sequence is added to the reaction mixture in the form of a template nucleic acid which is capable of homogeneous and continuous amplification and may be any intermediate in the cycle set forth in Figure 1. In particular, the template nucleic acid may be a single-stranded RNA which contains at its 5' end a sequence which is sufficiently homologous to that which is at the 3' end of the second primer, and contains a sequence at its 3' end which is sufficiently complementary to the first primer. A template nucleic acid of this form would function as a first template in the amplification process. Alternatively, the template nucleic acid may be a single-stranded DNA which contains at its 3' end a sequence which is sufficiently complementary to at least the 3' end of the second primer, and contains a sequence at its 5' end which is sufficiently homologous to that which is at the 3' end of the first primer. A template nucleic acid of this form would function as a 13408~'r second template in the amplification process.
Alternatively, the template nucleic acid may be a double-stranded DNA, one strand of which contains at its 5' end the entire sequence of the second primer and contains a sequence at its 3' end which is sufficiently complementary to the first primer. The double-stranded DNA functions as a third template in the amplification process.
Although the preparation of a template nucleic acid is not part of the amplification process, the description of possible schemes for generating template nucleic acids may be useful to the application of the amplification process. It should be understood that the schemes which may be used for obtaining the template nucleic acid are not limited to the alternatives which are described herein, and it is contemplated that other methods may be used.
In one alternative, a template nucleic acid which could function as a first template could be a naturally occurring RNA or a RNA fragment which could be generated from a larger RNA molecule by using site specific hydrolysis methods known in the art (Shibahara ~ al., 1987).
1340~~'~
In another alternative, a template nucleic acid which could function as a second template could be generated from a double-stranded DNA by digestion with a restriction endonuclease which has a site immediately flanking the sequence which is sufficiently complementary to the 3' end of the second primer. The resulting double-stranded DNA fragments could then be made single-stranded by using chemical or thermal denaturation methods.
In another alternative, a template nucleic acid which could function as a second template could be generated from a single-stranded DNA or RNA to which has been hybridized an oligonucleotide which is capable of blocking DNA synthesis. This blocking oligonucleotide may contain a chemical group, which could covalently bind to the template, under the appropriate conditions. DNA synthesis from this blocked template using the first primer could result in a synthesized DNA with the same 3' end as the second template. If the original template is RNA, then the resulting DNA-RNA hybrid may be used directly as a template nucleic acid. If the original template is DNA, then the resulting copy of the second template could then be separated from the original template by using chemical or thermal denaturation methods.
. 13~0~~'l In another alternative. a template nucleic acid which could function as a third template could be generated from a single-stranded DNA or RNA by DNA
synthesis from the DNA or RNA template using the second primer. The resulting synthesized DNA could then be separated from the original template by using chemical or thermal denaturation methods. In addition, a RNA template could be hydrolyzed by using chemical or enzymatic methods. The resulting single-stranded DNA has the_sequence of the second primer covalently attached to its 5' end and contains a sequence which is sufficiently complementary to the first primer. This single-stranded DNA could be converted to a transcriptionally functional double-stranded DNA by hybridizing the first primer to the single-stranded DNA, and by synthesizing a DNA
sequence which is covalently attached to the first primer and complementary to the single-stranded DNA.
In a further.alternative, a single-stranded DNA
or RNA template could be obtained from a double-stranded DNA, double-stranded RNA or a DNA-RNA
hybrid by using chemical, thermal, or possibly enzymatic methods. Then, by using one of the alternative schemes proposed above, the resulting iA
l~~Day~~
single-stranded DNA or RNA could then be used to generate a template nucleic acid which could function as a first, second or third template. In addition, an alternative scheme involving the first primer and one strand of nucleic acid, and another alternative scheme involving the second primer and the other (complementary) strand of the nucleic acid may be used concurrently to generate template nucleic acids.
MATERIALS AND METHODS
Materials Oligonucleotides were synthesized using an Applied Biosystems 380A DNA synthesizer. Columns, phosphoramidites, and reagents used for oligonucleotide synthesis were obtained from Applied Biosystems, Inc. through Technical Marketing Associates. Oligonucleotides were purified by polyacrylamide gel electrophoresis followed by DEAF
cellulose chromatography. The radioisotope [d~-32P]
UTP (800 Ci/mmol) was from Amersham. Enzymes for digesting and ligating DNA were purchased from New England Biolabs, and used according to the supplier's 13~OS~=~
recommendations. Preparations containing the large fragment of DNA polymerase 1 (Klenow) were also purchased from New England Biolabs. RNasin and T7 RNA
polymerase from Promega Biotec were purchased through Bio/Can Scientific Inc. Reverse transcriptase and RNase H were obtained from Pharmacia. The supplier for proteinase K was Boehringer Mannheim Canada. E.
coli strain HB101 (ATCC 33694) was used for all transformations. The plasmid pUCl9 (Norrander ~ al., 1983) was purchased from Bethesda Research Laboratories.
Isolation of DNA and sequencing E. coli transformants were grown on YT medium (Miller, 1972) containing 50 ug/ml ampicillin.
Plasmid DNA was purified by a rapid boiling method (Holmes and Quigley, 1981). DNA fragments and vectors used for all constructions were separated by electrophoresis on low melting point agarose, and purified from the molten agarose by phenol extraction and ethanol precipitation (Maniatis g~ al., 1982).
Plasmid DNA was sequenced using a modification (Hattori ~ ~. 1985) of the dideoxy method (Sanger g~ al., 1977). Reactions were run using the -20 universal primer (New England Biolabs).
1~~OSi~~
TCA precipitation Aliquots (5u1) of amplification reactions were quenched in 20u1 10 mM EDTA and placed on ice until all time point samples had been collected. The quenched samples were then applied to glass filter discs, and immediately dropped into ice-cold 5%
trichloroacetic acid ("TCA") - 1% sodium pyrophosphate for 10 min with occasional mixing. Two 5 min washes with ice-cold 5% TCA were followed by two additional washes with 95% ethanol and lyophilization to dryness. Radioactivity was determined in a liquid scintillation counter.
Polyacrylamide gel electrophoresis Samples (1 to 6 ul) were mixed with 4-5 ul formamide dye (90% deionized formamide, 10 mM TrisHCl (pH 8.0), 1 mM EDTA, xylene cyanol and bromophenol blue), and applied to a pre-run 12-cm- long 7%
denaturing polyacrylamide gel. Gels were run at 350 volts until the bromophenol blue dye had reached the bottom. In some cases the gels were fixed and dried prior to autoradiography. Fixing involved a 15 min wash in 10% methanol- 7% acetic acid. The profiles of ~~4~~~' the RNA products separated by this procedure were visualized by autoradiography at room temperature.
Example 1: Design and synthesis of oligonucleotides for a gag test system A synthetic DNA sequence (Figure 2A) was designed to include an EcoRI site, a T7 phage promoter, a sequence required for initiation of transcription by T7 RNA polymerase and a 19 by hybridization region (hybridization region 1). The 47 b antisense strand oligonucleotide (T7Hl.GAG) involved in the cloning of these elements also serves as the first primer.
Hybridization region 2 lies 53 by away from hybridization region 1 and is 20 by in length. The primer made to this region (H2.GAG) is a 20 b oligonucleotide duplicate of the sense strand and is not used for cloning. The sequence spanning and including the hybridization regions is a 92 by segment of the gag portion of the HIV-1 genome. This particular gene segment was chosen because the primers were predicted to hybridize efficiently and because the distance between the two hybridization regions was relatively short. In addition, a XbaI site was placed at the end of the sequence for cloning ease. The gag ~3~08~~
test sequence also contains SphI and PstI sites which can aid in the screening of recombinants.
A total of four oligonucleotides were used in the cloning of this fragment. N1.GAG, used in the construction of both the gag test and the gag2 test sequence, completes the antisense strand and is used only in the cloning process. Similarly, T74.PR0 is the sense strand component of the T7 promoter. N2.GAG
however, was used in the construction of both test fragments, and has also been used as an intermediate (second template) in two steps of the amplification cycle. The entire cloned gag test fragment can also represent an intermediate of the amplification cycle (third template). Once cloned into an appropriate vector the gag test DNA could be transcribed by T7 RNA
polymerase to produce a RNA fragment (first template) useful as an amplification intermediate involved in three of the steps. In addition, T7H1.GAG and H2.GAG
serve as primers in the test system.
The gag2 test synthetic DNA fragment (Figure 2B) does not contain the T7 promoter, but the remainder of the sequence is identical to the gag test sequence and therefore, both N1.GAG and N2.GAG were involved in its x ;~
'~ v..
134U8~' construction. The oligonucleotide required to complete the antisense strand is called H1.GAG. Once cloned, the gag2 test fragment can be used as a template for testing amplification, using a DNA
restriction fragment as template nucleic acid.
Example 2: Construction of the gag test plasmids The oligonucleotides T74.PR0 and N1.GAG (2 ug each), were phosphorylated separately in 20 ul reactions containing 70 mM TrisHCl (pH 7.6), 10 mM
MgCl2, 5 mM DTT, 0.5 mM ATP and 5 units T4 polynucleotide kinase, at 37°C for 30 min.
Phosphorylated T74.PR0 and N1.GAG (10 ul of each) were mixed with 1 ug each of unphosphorylated T7Hl.GAG and N2.GAG, and 3 ul 100 mM TrisHCl (pH7.8) - 500 mM NaCl, in a final volume of 29 ul for the gag test assembly.
The gag2 test mixture contained 10 ul phosphorylated N1.GAG, 1 ug each of unphosphorylated H1.GAG and N2.GAG, and 1.8 ul 100 mM TrisHCl (pH 7.8) - 500 mM
NaCl, in a final volume of 18 ul. The oligonucleotide mixtures were hybridized separately by placing them at 90°C for 10 min followed by slow cooling to room temperature for 10 to 16 h 60 ul reactions containing 50 mM TrisHCl (pH 7.8), 10 mM MgCl2, 20 mM DTT, 1mM
13~0~~'I
ATP and 50 ug/ml BSA were used to ligate the hybridized oligonucleotides together. 400 units T4 DNA ligase was added to the gag test reaction and it was incubated at 15°C for 2h while the gag2 test reaction was incubated for 14 to 16 h with 200 units T4 DNA ligase.
The isolated and purified synthetic DNA segments were mixed with plasmid pUCl9 which had been linearized by digestion at restriction enzyme sites within the polylinker region. T4 DNA ligase was used to ligate the gag test sequence into the EcoRI- XbaI
fragment of pUCl9, while the gag2 test sequence was ligated to the SmaI-XbaI fragment. Plasmid DNA from transformants obtained after these reactions were used to transform E. coli were screened by restriction analysis , and the final plasmids (pGAG.TEST and pGAG2.TEST) were determined to be correct by sequence analysis.
Example 3: Effect of primer concentration on RNA
amplification The reaction mixtures (25 ul) which were used to amplify RNA transcribed from the gag test ,..Y I
1340~0'r oligonucleotides contained 50 nM TrisHC1 (pH
8.45), 6 mM MgCl2 40 mM KC1, 10 mM
dithiothreitol, 0.5 mM NTP (ATP, CTP, GTP, UTP), 1 mM dNTP (dATP, dCTP, dGTP, dTTP), 20 units RNasin, 10 units T7 RNA polymerase, 10 units reverse transcriptase, 0.4 units RNase H, and 10 uCi [ a -32P] UTP. Two of the reactions contained 0.5 ng (0.015 pmoles) N2.GAG while the other two reactions contained no template. The primers T7Hl.GAG and H2.GAG were each added at final concentrations of 3.4 uM or 0.34 uM to reactions containing either N2.GAG or no template. The reactions were incubated at 42°C
for 2h. Total synthesis of RNA was monitored by determining the incorporation of TCA insoluble cpm at 30 min. intervals. The effect of the primer concentration on template-dependent RNA
synthesis is shown in Table 1. Aliquots of each reaction, containing equal amounts of synthesized RNA, were analyzed by PAGE and autoradiography (Figure 3, lanes 1-4 numbered the same as the reactions).
Table 1: RNA amplification after 2 h.
Concentration of Template RNA
Reaction each primier (ng) Synthesized (uM) (ug) 1 3.4 0.5 2.8 2 3.4 - 2.1 3 0.34 0.5 1.8 4 0.34 - 0,-7 13408i~'7 It was found that while reaction 1 resulted in the greatest incorporation of isotope, the no template control, reaction 2, was also high (73% of reaction 1) and produced a very similar electrophoretic profile.
It would therefore appear that in the presence of high primer concentrations, a RNA transcript of identical size to that expected in amplification is produced in the absence of any template. Results using samples with a 10- fold decrease in primer concentration were dramatically different. The amount of RNA produced in reaction 3 was 2.6 times that of reaction 4, but while virtually all of the transcript was found in a single band of the expected size in reaction 3, no fragments greater than 60 to 70 b were found in reaction 4.
Primer concentration therefore plays a significant role in the accuracy and efficiency of RNA
amplification.
A control RNA transcript used to represent the size of fragment expected to be generated by the amplification system (lane 0 of Figure 3) was prepared by transcription from the test plasmid. pGAG.TEST was linearized by digestion with XbaI, proteinase K
treated (Maniatis g~ al., 1982), phenol extracted and ethanol precipitated. T7 RNA polymerase was then used 134~8~'~
according to the supplier's recommendations to transcribe 0.5 ug of the resulting fragment in a 25 ul reaction mixture containing 10 uCi [ a' -32P] UTP.
example 4: Effect of template concentration on RNA
amplification The standard 50 ul reaction mixture used to amplify RNA transcribed from the gag test oligonucleotides contained 0.34 uM T7H1.GAG, 0.34 uM
H2.GAG, 50 mM TrisHCl (pH 8.45), 6 mM MgCl2, 40 mM
KC1, 10 mM DTT, 0.5 mM NTP, 1 mM dNTP, 40 units RNasin, 20 units T7 RNA polymerase, 20 units reverse transcriptase, 0.8 units RNase H and 10 - 20 uCi [ ~-32P] UTP. The reactions contained amounts of template (N2.GAG) varying from 1 ng to 1 fg. One reaction contained no template. The reactions were incubated at 42°C for 3h, during which total synthesis of RNA was monitored by determining the incorporation of TCA insoluble cpm at 30 min intervals. As indicated in Table 2, total RNA synthesis was higher than the no template control for all template concentrations tested. Although the total synthesis of RNA generally decreased with decreasing template concentration, this decrease in synthesis was not Z~40~0'l quantitative. Thus, the degree of amplification of RNA per starting template generally increased with decreasing template concentration. Amplification of 8 x 108 fold was achieved by synthesis of 0.8 ug RNA
from 1 fg of N2.GAG template. One fg of the 102 b N2.GAG oligonucleotide represents approximately 2 a 104 molecules.
Table 2: RNA amplification from N2.GAG after 3h.
RNA
Synthesized Fold Reaction Template (ug) amplifi cation 1 1 ng 3.5 3.5 x 103 2 100 pg 4.4 4.4 x 104 3 10 pg 4.1 4.1 x 105 4 1 pg 3.0 3.0 x 106 5 100 fg 2.7 2.7 x 107 6 10 fg 1.9 1.9 x 108 7 1 fg 0.78 7.8 x 108 8 - 0Ø46 -The RNA which was synthesized after a reaction time of 3h was analyzed by PAGE, for each template concentration (Figure 4 lanes 1-8, numbered the same as the reactions). A major band representing a RNA of about 100 b was present in all reactions except the ones containing 1 fg template and no template. The reaction containing 1 fg template did not have much of this 100 b product at 3 h but the overall RNA
.1~~C~~i~'l synthesis was higher than and qualitatively different from the no template reaction.
Example 5: Hybridization analysis of RNA products.
Amplification reactions containing amounts of N2.GAG template varying from 1 pg to 0.1 fg were performed following the teaching of example 4, except the radiolabeled UTP was omitted. The reactions were incubated at 42°C for 3 h. Aliquots were removed from each reaction at 30 min intervals and applied to a nylon membrane (Amersham). The nucleic acids that were contained in these reaction aliquots were fixed by exposure to ultraviolet light. The membrane was prehybridized at 50°C for 1 h in prehybridization buffer consisting of a final concentration of 50% v/v formamide, 5 X SSC and 5 X Denhardt's solution (Maniatis ~ ~. 1982; Southern g~ al., 1975) at a volume equivalent to 5 mls of solution per 100 cm2 and hybridized with a radiolabeled probe with a specific activity of 106 cpm/ml of hybridization solution. Hybridization was performed at 50°C for 16 h in 50% formamide, 5 X SSC and 5 X Denhardt's solution (Maniatis g~ ~. 1982; Southern g~ al., 1975). The radiolabeled probe was the synthetic ~3408J7 oligonucleotide 5' GATCTGGGATAGAGTACATCCA 3' which had been labeled at the 5' end using T4 polynucleotide kinase and ( Y - 3zP) ATP. After the membrane was washed at 50°C in a series of 2, 3 min. washes consisting of 2 X SSC, 0.1$ v/v SDS and 0.2 X SCC, 0.1~ v/v SDS (Southern et al., 1975; Maniatis et al., 1982; Szostak et al., 1979).
Figure 5 shows the results of the hybridization analysis performed on the amplification reactions, containing various amounts of N2.GAG template, which were sampled at different times of incubation.
Each column of Figure 5 represents a different time point (l, 30 min; 2, 60 min; 3, 90 min; 4, 120 min; 5, 150 min; 6, 180 min) and each row represents a different amount of added N2.GAG template (1, lpg; 2, 100 fg; 3, 10 fg; 4, 1 fg; 5, 0.1 fg; 6, no template). Amplification of nucleic acids which hybridized to the labeled probe were observed for rows 1-3 (1 pg - 10 fg), however the hybridization to specific nucleic acids in rows 4 - 5 (1 fg, 0.1 fg) was not higher than row 6 (no template). The apparent non-specific binding of labeled probe in row 6 seems to be related to DNA or RNA synthesis since the hybridization signal increases with increasing time.
.-13~U8~~
Example 6: Use of DNA restriction fragment as template.
The plasmid pGAG2.TEST was digested with MspI and proteinase K, purified by phenol extraction and ethanol precipitation, and denatured by boiling for 5 min. Amplification reactions were performed and analyzed following the teaching of Example 4, except the MspI digested pGAG2.TEST was used as a template instead of the N2.GAG oligonucleotide. The amounts of plasmid added to each reaction varied from 55 ng to 5.5 pg, and no template. To simulate additional DNA
which would be present in an actual sample, alternate reactions contained 1 ng of calf thymus DNA which had been similarly digested, purified and denatured.
After a 3 h incubation at 42°C, the synthesis of RNA
was determined by TCA precipitation and PAGE
analysis. As indicated in Table 3, total RNA
synthesis was higher than the no template controls for all template concentrations tested. The degree of amplification was calculated based on RNA synthesis from the actual template which was 1.8% of the total plasmid DNA.
The total RNA synthesis (degree of amplification) from a particular initial level template concentration ~r~, m:,, 1340~0'~
was consistently lower for the restriction.fragment (Table 3) as compared to that for the synthetic oligonucleotide template (Table 2). This could be due to competition with the complementary strand of the restriction fragment template under the conditions used.
Table 3: RNA amplification from MspI-digested pGAG2.TEST
RNA Fold Reaction Template* Synth esized** amplific ation**
1 55.0 ng [ 1 ng] 3.65 3.7 x 103 2 (4.05) (4.1 x 103) 3 5.5 ng [100 pg] 3.54 3.5 x 104 4 (3.16) (3.2 x 104) 5 550.0 pg [ 10 pg] 2.29 2.3 x 105 6 (2.79) (2.8 x 105) 7 55.0 pg [ 1 pg] 2.62 2.6 x 106 8 (0.67) (0.7 x 106) 9 5.5 pg [100 fg] 1.37 1.4 x 107 10 (2.26) (2.3 x 107) 11 - 1.25 -12 (0.08) 2 0 * Numbersis brackets indicate valent amountsof GAG.
equi N2.
** Numbers in parentheses indicateRNA synthesisin ce of presen 1 ug MspI-di gested calf thymus DNA.
,. ~ 1340~i~'~
The RNA which was synthesized after a reaction time of 3h was analyzed by PAGE (Figure 6, lanes 1-6, 11 and 12, numbered the same as the reactions). The major band representing a RNA of about 100 b was present in reactions (lanes) 1-6 but absent in the no template reactions (lanes 11 and 12). The RNA in lane 0 was a standard, which was prepared following the teaching of Example 3. There was no apparent qualitative difference in the synthesized RNA either with (lanes 2, 4 and 6) or without (lanes 1, 3, and 5) the additional of 1 ~g of MspI-digested calf thymus DNA.
Although preferred embodiments of the invention have been described in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from either the spirit of the invention or the scope of the appended claims.
~.. 1340~i~r~
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Claims (44)
1. A process for the amplification of a specific nucleic acid sequence, at a relatively constant temperature and without serial addition of reagents, comprising the steps of:
(A) providing a single reaction medium containing reagents comprising (i) a first oligonucleotide primer;
(ii) a second oligonucleotide primer comprising an antisense sequence of a promoter recognized by an RNA polymerase;
(iii) a DNA-directed RNA polymerase that recognizes said promoter;
(iv) an RNA-directed DNA polymerase;
(v) a DNA-directed DNA polymerase;
(vi) a ribonuclease that hydrolyzes RNA of an RNA-DNA hybrid without hydrolyzing single- or double-stranded RNA or DNA, and (vii) ribonucleoside and deoxyribonucleoside triphosphates;
then (B) providing in said reaction medium RNA comprising an RNA first template which comprises said specific nucleic acid sequence or a sequence complementary to said specific nucleic acid sequence, under conditions such that a cycle ensues wherein (i) said first oligonucleotide primer hybridizes to said RNA first template;
(ii) said RNA-directed DNA polymerase uses said RNA first template to synthesize a DNA
second template by extension of said first oligonucleotide primer and thereby forms an RNA-DNA hybrid intermediate:
(iii) said ribonuclease hydrolyzes RNA which comprises said RNA-DNA hybrid intermediate;
(iv) said second oligonucleotide primer hybridizes to said DNA second template;
(v) said DNA-directed DNA polymerase uses said second oligonucleotide primer as a template to synthesize a functional promoter recognized by said RNA polymerase by extension of said DNA second template;
(vi) said DNA-directed RNA polymerase recognizes said functional promoter and transcribes said DNA second template, thereby providing copies of said RNA first template;
and thereafter (C) maintaining said conditions for a time sufficient to achieve a desired amplification of said specific nucleic acid sequence.
(A) providing a single reaction medium containing reagents comprising (i) a first oligonucleotide primer;
(ii) a second oligonucleotide primer comprising an antisense sequence of a promoter recognized by an RNA polymerase;
(iii) a DNA-directed RNA polymerase that recognizes said promoter;
(iv) an RNA-directed DNA polymerase;
(v) a DNA-directed DNA polymerase;
(vi) a ribonuclease that hydrolyzes RNA of an RNA-DNA hybrid without hydrolyzing single- or double-stranded RNA or DNA, and (vii) ribonucleoside and deoxyribonucleoside triphosphates;
then (B) providing in said reaction medium RNA comprising an RNA first template which comprises said specific nucleic acid sequence or a sequence complementary to said specific nucleic acid sequence, under conditions such that a cycle ensues wherein (i) said first oligonucleotide primer hybridizes to said RNA first template;
(ii) said RNA-directed DNA polymerase uses said RNA first template to synthesize a DNA
second template by extension of said first oligonucleotide primer and thereby forms an RNA-DNA hybrid intermediate:
(iii) said ribonuclease hydrolyzes RNA which comprises said RNA-DNA hybrid intermediate;
(iv) said second oligonucleotide primer hybridizes to said DNA second template;
(v) said DNA-directed DNA polymerase uses said second oligonucleotide primer as a template to synthesize a functional promoter recognized by said RNA polymerase by extension of said DNA second template;
(vi) said DNA-directed RNA polymerase recognizes said functional promoter and transcribes said DNA second template, thereby providing copies of said RNA first template;
and thereafter (C) maintaining said conditions for a time sufficient to achieve a desired amplification of said specific nucleic acid sequence.
2. A process according to claim 1, wherein said RNA
first template comprises said specific nucleic acid sequence and wherein step (B) comprises providing single-stranded RNA to said reaction medium such that (i) said first oligonucleotide primer hybridizes to said single-stranded RNA;
(ii) said RNA-directed DNA polymerase uses said single-stranded RNA as a template to synthesize a DNA second template by extension of said first oligonucleotide primer and thereby forms an RNA-DNA hybrid;
(iii) said ribonuclease hydrolyzes RNA which comprises said RNA-DNA hybrid;
(iv) said second oligonucleotide primer hybridizes to said DNA second template;
(v) said DNA-directed DNA polymerase uses said second oligonucleotide primer as template to synthesize a functional promoter recognized by said DNA-directed RNA polymerase by extension of said DNA second template;
and (vi) said DNA-directed RNA polymerase recognizes said functional promoter and transcribes said DNA
second template, thereby providing copies of said RNA first template.
first template comprises said specific nucleic acid sequence and wherein step (B) comprises providing single-stranded RNA to said reaction medium such that (i) said first oligonucleotide primer hybridizes to said single-stranded RNA;
(ii) said RNA-directed DNA polymerase uses said single-stranded RNA as a template to synthesize a DNA second template by extension of said first oligonucleotide primer and thereby forms an RNA-DNA hybrid;
(iii) said ribonuclease hydrolyzes RNA which comprises said RNA-DNA hybrid;
(iv) said second oligonucleotide primer hybridizes to said DNA second template;
(v) said DNA-directed DNA polymerase uses said second oligonucleotide primer as template to synthesize a functional promoter recognized by said DNA-directed RNA polymerase by extension of said DNA second template;
and (vi) said DNA-directed RNA polymerase recognizes said functional promoter and transcribes said DNA
second template, thereby providing copies of said RNA first template.
3. A process according to claim 2, wherein step (B) comprises adding to said reaction medium single-stranded RNA.
4. A process according to claim 1, wherein said RNA
first template comprises a sequence complementary to said specific nucleic acid sequence and wherein step (B) comprises adding single-stranded RNA to said reaction medium such that (i) said second oligonucleotide primer hybridizes to said single-stranded RNA;
(ii) said RNA-directed DNA polymerase uses said RNA
as a template to synthesize a complementary DNA
by extension of said second oligonucleotide primer and thereby forms an RNA-DNA hybrid;
(iii) said ribonuclease hydrolyzes RNA which comprises said RNA-DNA hybrid;
(iv) said first oligonucleotide primer hybridizes to said complementary DNA;
(v) said DNA-directed DNA polymerase uses said complementary DNA as template to synthesize said DNA second template and a functional promoter recognized by said RNA polymerase by extension of said first oligonucleotide primer;
and (vi) said DNA-directed RNA polymerase recognizes said functional promoter and transcribes said DNA
second template thereby providing copies of said RNA first template.
first template comprises a sequence complementary to said specific nucleic acid sequence and wherein step (B) comprises adding single-stranded RNA to said reaction medium such that (i) said second oligonucleotide primer hybridizes to said single-stranded RNA;
(ii) said RNA-directed DNA polymerase uses said RNA
as a template to synthesize a complementary DNA
by extension of said second oligonucleotide primer and thereby forms an RNA-DNA hybrid;
(iii) said ribonuclease hydrolyzes RNA which comprises said RNA-DNA hybrid;
(iv) said first oligonucleotide primer hybridizes to said complementary DNA;
(v) said DNA-directed DNA polymerase uses said complementary DNA as template to synthesize said DNA second template and a functional promoter recognized by said RNA polymerase by extension of said first oligonucleotide primer;
and (vi) said DNA-directed RNA polymerase recognizes said functional promoter and transcribes said DNA
second template thereby providing copies of said RNA first template.
5. A process according to claim 4, wherein step (B) comprises adding to said reaction medium said single-stranded RNA.
6. A process according to claim 1, wherein step (B) comprises adding to said reaction medium single-stranded DNA which comprises an antisense sequence of a promoter recognized by said RNA
polymerase, such that (i) said first oligonucleotide primer hybridizes to said single-stranded DNA;
(ii) said DNA-directed DNA polymerase uses said single-stranded DNA as template to synthesize said DNA second template and a functional promoter recognized by said DNA-directed RNA
polymerase by extension of said first oligonucleotide primer;
and (iii) said DNA-directed RNA polymerase recognizes said functional promoter and transcribes said DNA
second template, thereby providing copies of said RNA first template.
polymerase, such that (i) said first oligonucleotide primer hybridizes to said single-stranded DNA;
(ii) said DNA-directed DNA polymerase uses said single-stranded DNA as template to synthesize said DNA second template and a functional promoter recognized by said DNA-directed RNA
polymerase by extension of said first oligonucleotide primer;
and (iii) said DNA-directed RNA polymerase recognizes said functional promoter and transcribes said DNA
second template, thereby providing copies of said RNA first template.
7. A process according to claim 6, wherein step (B) comprises adding to said reaction medium an RNA-DNA
hybrid comprising said single-stranded DNA, such that said ribonuclease hydrolyzes RNA which comprises said RNA-DNA hybrid.
hybrid comprising said single-stranded DNA, such that said ribonuclease hydrolyzes RNA which comprises said RNA-DNA hybrid.
8. A process according to claim 1, wherein step (B) comprises adding to said reaction medium single-stranded DNA which comprises said DNA second template, such that (i) said second oligonucleotide primer hybridizes to said single-stranded DNA;
(ii) said DNA-directed DNA polymerase uses said second oligonucleotide primer as template to synthesize a functional promoter recognized by said DNA-directed RNA polymerase by extension of said DNA second template;
and (iii) said DNA-directed RNA polymerase recognizes said functional promoter and transcribes said DNA
second template, thereby providing copies of said RNA first template.
(ii) said DNA-directed DNA polymerase uses said second oligonucleotide primer as template to synthesize a functional promoter recognized by said DNA-directed RNA polymerase by extension of said DNA second template;
and (iii) said DNA-directed RNA polymerase recognizes said functional promoter and transcribes said DNA
second template, thereby providing copies of said RNA first template.
9. A process according to claim 8, wherein step (B) comprises adding to said reaction medium an RNA-DNA
hybrid comprising said single-stranded DNA, such that said ribonuclease hydrolyzes RNA which comprises said RNA-DNA hybrid.
hybrid comprising said single-stranded DNA, such that said ribonuclease hydrolyzes RNA which comprises said RNA-DNA hybrid.
10. A process according to claim 2, wherein step (B) comprises adding to said reaction medium a DNA
comprising a functional promoter recognized by said RNA polymerase, such that said DNA-directed RNA
polymerase transcribes said DNA, thereby synthesizing said single-stranded RNA.
comprising a functional promoter recognized by said RNA polymerase, such that said DNA-directed RNA
polymerase transcribes said DNA, thereby synthesizing said single-stranded RNA.
11. A process according to claim 4, wherein step (B) comprises adding to said reaction medium a DNA
comprising a functional promoter recognized by said RNA polymerase, such that said DNA-directed RNA
polymerase transcribes said DNA, thereby synthesizing said single-stranded RNA.
comprising a functional promoter recognized by said RNA polymerase, such that said DNA-directed RNA
polymerase transcribes said DNA, thereby synthesizing said single-stranded RNA.
12. A process according to claim 1, wherein said second oligonucleotide primer further comprises an antisense sequence of a transcription initiation site for said DNA-directed RNA polymerase, said antisense sequence of said transcription initiation site being operatively linked to said antisense sequence of said promoter.
13. A process according to claim 12, wherein said DNA-directed RNA polymerase is bacteriophage T7 RNA
polymerase and wherein said antisense sequence of a transcription initiation site and said antisense sequence of said promoter together comprise the nucleotide sequence:
AATTCTAATACGACTCACTATAGGGAG.
polymerase and wherein said antisense sequence of a transcription initiation site and said antisense sequence of said promoter together comprise the nucleotide sequence:
AATTCTAATACGACTCACTATAGGGAG.
14. A process according to claim 1, wherein step (B) further comprises adding a sample to said reaction medium under conditions such that, if said sample thereby provides RNA comprising an RNA first template which comprises said specific nucleic acid sequence or a sequence complementary to said specific nucleic acid sequence, said cycle ensues, and wherein said process further comprises, after step (C), a step (D) of monitoring said reaction medium for consumption of any of said reagents (i), (ii) and (vii) or for accumulation of any product of said cycle.
15. A process according to claim 14, wherein step (D) comprises detecting a nucleic acid product of said cycle.
16. A process according to claim 15, wherein step (D) comprises detecting said nucleic acid product using a nucleic acid probe.
17. A process according to claim 15, wherein step (D) comprises detecting said nucleic acid product using restriction endonucleases and electrophoretic separation.
18. A process according to claim 15, wherein step (D) comprises monitoring the accumulation of said RNA
first template.
first template.
19. A process according to claim 15, wherein step (D) comprises monitoring the accumulation of said DNA
second template.
second template.
20. A process according to claim 15, wherein step (D) comprises monitoring DNA containing a functional promoter recognized by said RNA polymerase.
21. A process according to claim 15, wherein step (D) comprises monitoring the accumulation of said RNA-DNA
hybrid intermediate.
hybrid intermediate.
22. A process according to claim 14, wherein step (D) further comprises comparing consumption of any reagent of said reagents (i), (ii) and (vii) of step (A) or accumulation of any product of said cycle with a value representing consumption of said reagent or accumulation of said product in said reaction medium in the absence of said specific nucleic acid sequence and said sequence complementary thereto.
23. A process according to claim 1, wherein said ribonuclease comprises Escherichia coli ribonuclease H.
24. A process according to claim 1, wherein said ribonuclease comprises calf thymus ribonuclease H.
25. A process according to claim 1, wherein said first oligonucleotide primer or said second oligonucleotide primer is bound reversibly to an immobilized support.
26. A process according to claim 1, wherein said DNA-directed RNA polymerase is a bacteriophage RNA
polymerase.
polymerase.
27. A process according to claim 26, wherein said DNA-directed RNA polymerase is bacteriophage T7 RNA
polymerase.
polymerase.
28. A process according to claim 26, wherein said DNA-directed RNA polymerase is bacteriophage T3 polymerase.
29. A process according to claim 26, wherein said DNA-directed RNA polymerase is bacteriophage ~II
polymerase.
polymerase.
30. A process according to claim 26, wherein said DNA-directed RNA polymerase is Salmonella bacteriophage sp6 polymerase.
31. A process according to claim 26, wherein said DNA-directed RNA polymerase is Pseudomonas bacteriophage gh-1 polymerase.
32. A process according to claim 1, wherein said RNA-directed DNA polymerase is a retroviral reverse transcriptase.
33. A process according to claim 32, wherein the said retroviral reverse transcriptase is avian myeloblastosis virus reverse transcriptase.
34. A process according to claim 32, wherein said retrovirus reverse transcriptase is a Moloney murine leukemia virus reverse transcriptase.
35. A process according to claim 1, wherein said DNA-directed RNA polymerase lacks exonuclease activity.
36. A process according to claim 1, wherein all DNA
polymerases in said reaction medium lack DNA
exonuclease and endonuclease activity.
polymerases in said reaction medium lack DNA
exonuclease and endonuclease activity.
37. A process according to claim 1, wherein said DNA-directed DNA polymerase is avian myeloblastosis virus polymerase.
38. A process according to claim 1, wherein said DNA-directed DNA polymerase is DNA polymerase ~ or .beta..
39. A process according to claim 1, wherein said DNA-directed DNA polymerase is calf thymus DNA
polymerase.
polymerase.
40. A process according to claim 1, wherein step (C) comprises maintaining said conditions for a period of time between 30 minutes and 3 hours.
41. A process according to claim 1, further comprising the steps of ligating a DNA product of said cycle into a cloning vector and then cloning said DNA
product.
product.
42. A process according to claim 41, further comprising the step of expressing a product encoded by said DNA
product of said cycle in an expression system.
product of said cycle in an expression system.
43. A kit for amplifying nucleic acid molecules, comprising an assemblage of:
(A) a first oligonucleotide primer having a DNA
sequence sufficiently complementary to an RNA
sequence of a first template to be amplified;
(B) a second oligonucleotide primer comprising an antisense sequence of a promoter recognized as a substrate for an RNA polymerase and having a DNA
sequence sufficiently complementary to a DNA
sequence of a second template;
(C) a ribonuclease that hydrolyzes RNA of an RNA/DNA
hybrid without attacking single-stranded or double-stranded RNA or DNA;
(D) an RNA-directed DNA polymerase;
(E) DNA-directed DNA polymerase;
(F) a DNA-directed RNA polymerase;
(G) ribonucleoside triphosphates; and (H) deoxyribonucleoside triphosphates;
where the DNA sequence of the second template is complementary to the RNA sequence of the first template.
(A) a first oligonucleotide primer having a DNA
sequence sufficiently complementary to an RNA
sequence of a first template to be amplified;
(B) a second oligonucleotide primer comprising an antisense sequence of a promoter recognized as a substrate for an RNA polymerase and having a DNA
sequence sufficiently complementary to a DNA
sequence of a second template;
(C) a ribonuclease that hydrolyzes RNA of an RNA/DNA
hybrid without attacking single-stranded or double-stranded RNA or DNA;
(D) an RNA-directed DNA polymerase;
(E) DNA-directed DNA polymerase;
(F) a DNA-directed RNA polymerase;
(G) ribonucleoside triphosphates; and (H) deoxyribonucleoside triphosphates;
where the DNA sequence of the second template is complementary to the RNA sequence of the first template.
44. A kit for amplifying a specific nucleic acid molecules comprising:
(A) a first oligonucleotide primer having a DNA
sequence sufficiently complementary to an RNA
sequence of a first template to be amplified;
(B) a second oligonucleotide primer comprising an antisense sequence of a promoter recognized as a substrate for an RNA polymerase and having a DNA
sequence sufficiently complementary to a DNA
sequence of a second template;
(C) Escherichia coli ribonuclease H;
(D) avian myeloblastosis virus reverse transcriptase;
(E) bacteriophage T7 RNA polymerase;
(F) ribonucleoside triphosphates; and (G) deoxyribonucleoside triphosphates;
where the DNA sequence of the second template is complementary to the RNA sequence of the first template.
(A) a first oligonucleotide primer having a DNA
sequence sufficiently complementary to an RNA
sequence of a first template to be amplified;
(B) a second oligonucleotide primer comprising an antisense sequence of a promoter recognized as a substrate for an RNA polymerase and having a DNA
sequence sufficiently complementary to a DNA
sequence of a second template;
(C) Escherichia coli ribonuclease H;
(D) avian myeloblastosis virus reverse transcriptase;
(E) bacteriophage T7 RNA polymerase;
(F) ribonucleoside triphosphates; and (G) deoxyribonucleoside triphosphates;
where the DNA sequence of the second template is complementary to the RNA sequence of the first template.
Priority Applications (13)
Application Number | Priority Date | Filing Date | Title |
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CA000559709A CA1340807C (en) | 1988-02-24 | 1988-02-24 | Nucleic acid amplification process |
US07/211,384 US5409818A (en) | 1988-02-24 | 1988-06-24 | Nucleic acid amplification process |
ES88113948T ES2053648T3 (en) | 1988-02-24 | 1988-08-26 | PROCESS OF EXPANSION OF A NUCLEIC ACID. |
AT88113948T ATE106948T1 (en) | 1988-02-24 | 1988-08-26 | METHODS FOR AMPLIFICATION OF NUCLEIC ACIDS. |
EP88113948A EP0329822B1 (en) | 1988-02-24 | 1988-08-26 | Nucleic acid amplification process |
DE3850093T DE3850093T2 (en) | 1988-02-24 | 1988-08-26 | Methods for amplifying nucleic acids. |
JP1014934A JP2650159B2 (en) | 1988-02-24 | 1989-01-24 | Nucleic acid amplification method |
KR1019890002142A KR960015744B1 (en) | 1988-02-24 | 1989-02-23 | Nucleic acid amplication process |
KR1019910700397A KR920702866A (en) | 1988-02-24 | 1989-08-19 | Nucleic Acid Amplification Method |
PCT/EP1989/000981 WO1991002814A1 (en) | 1988-02-24 | 1989-08-19 | Nucleic acid amplification process |
US08/386,774 US5554517A (en) | 1988-02-24 | 1995-02-10 | Nucleic acid amplification process |
US08/606,817 US6063603A (en) | 1988-02-24 | 1996-02-26 | Nucleic acid amplification process |
JP9057599A JPH09327298A (en) | 1988-02-24 | 1997-03-12 | Amplification of nucleic acid |
Applications Claiming Priority (1)
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CA000559709A CA1340807C (en) | 1988-02-24 | 1988-02-24 | Nucleic acid amplification process |
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US (1) | US5409818A (en) |
EP (1) | EP0329822B1 (en) |
KR (2) | KR960015744B1 (en) |
AT (1) | ATE106948T1 (en) |
CA (1) | CA1340807C (en) |
DE (1) | DE3850093T2 (en) |
ES (1) | ES2053648T3 (en) |
WO (1) | WO1991002814A1 (en) |
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US8044184B2 (en) | 2004-04-26 | 2011-10-25 | Wako Pure Chemical Industries, Ltd. | Probe and primer for tubercle bacillus detection, and method of detecting human tubercle bacillus therewith |
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JP2650159B2 (en) * | 1988-02-24 | 1997-09-03 | アクゾ・ノベル・エヌ・ベー | Nucleic acid amplification method |
CA1340807C (en) * | 1988-02-24 | 1999-11-02 | Lawrence T. Malek | Nucleic acid amplification process |
US5130238A (en) * | 1988-06-24 | 1992-07-14 | Cangene Corporation | Enhanced nucleic acid amplification process |
AU633036B2 (en) * | 1988-12-09 | 1993-01-21 | Chemicon International, Inc. | Amplified dna assay |
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1989
- 1989-02-23 KR KR1019890002142A patent/KR960015744B1/en not_active IP Right Cessation
- 1989-08-19 WO PCT/EP1989/000981 patent/WO1991002814A1/en active Application Filing
- 1989-08-19 KR KR1019910700397A patent/KR920702866A/en not_active Application Discontinuation
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8044184B2 (en) | 2004-04-26 | 2011-10-25 | Wako Pure Chemical Industries, Ltd. | Probe and primer for tubercle bacillus detection, and method of detecting human tubercle bacillus therewith |
US8628926B2 (en) | 2004-04-26 | 2014-01-14 | Wako Pure Chemical Industries, Ltd. | Probe and primer for tubercle bacillus detection, and method of detecting human tubercle bacillus therewith |
Also Published As
Publication number | Publication date |
---|---|
EP0329822B1 (en) | 1994-06-08 |
KR890013184A (en) | 1989-09-21 |
KR920702866A (en) | 1992-10-28 |
ES2053648T3 (en) | 1994-08-01 |
DE3850093D1 (en) | 1994-07-14 |
KR960015744B1 (en) | 1996-11-20 |
US5409818A (en) | 1995-04-25 |
DE3850093T2 (en) | 1994-11-03 |
ATE106948T1 (en) | 1994-06-15 |
EP0329822A2 (en) | 1989-08-30 |
EP0329822A3 (en) | 1990-08-01 |
WO1991002814A1 (en) | 1991-03-07 |
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