CA2102963A1 - Simple nucleic acid amplification - Google Patents
Simple nucleic acid amplificationInfo
- Publication number
- CA2102963A1 CA2102963A1 CA002102963A CA2102963A CA2102963A1 CA 2102963 A1 CA2102963 A1 CA 2102963A1 CA 002102963 A CA002102963 A CA 002102963A CA 2102963 A CA2102963 A CA 2102963A CA 2102963 A1 CA2102963 A1 CA 2102963A1
- Authority
- CA
- Canada
- Prior art keywords
- strand
- primer
- nucleic acid
- sequence
- promoter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
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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]
Abstract
Abstract Process for amplifying deoxyribonucleic acids according to the principle of a transcription amplification, which does not require separate cDNA formation.
Description
~-`` 21~2963 Simple nucleic acid am~lification ~ocess The subject matter of the invention includes a process for amplifying a segment of a deoxyI~bonucleic acid, a process for detecting this deoxyribonucleic acid, and reagents for the implementation of said processes.
Samples, such as body fluids, contain deoxyribonucleic acids only in minute amounts. These contain, however, sequence information which is often a fairly reliable indicator for the presence of certain organisms or the conditions of such organisms. Amplifying the sequence info~nation has proven to be a usefill tool. While the first applicable amplification systems were based on ~e exponential amplification of sequence information in temperature cycles, more recent systems allow these reactions to occur at almost constant temperatures.
Such a system is described in EP-A-O 329 822, for example. This publication describes a process for amplifying ribonucleic acids where an antisense-p~omoter-containing oligonucleotide is hybridized to the one end of the nucleic acid to be amplified. Using the nucleic acid as a template, this oligonucleotide is extended by adding deoxyTibonucleotides. After the RNA/DNA hybrid is denatured, another oligonucleotide is hybridized to the end of the DNA which is remote from the promoter sequence and is extended under the formation of a functional promoter.
The segment of the double strand which follows the promoter is then transcribed into RNA under control of the promoter. Then, the RNA is again used to synthesize the promoter-containing DNA double strand. A great disadvantage of this process is that if DNA is used as the initial template, thecorresponding sequence is either chemically or synthetically produced or cleaved out of a plasmid by means of a restriction enzyme such ~at the nucleotides of the nucleic acid to be amplified which are located at the 5'-end hybridi~e to the promoter oligonucleotide. Yet another disadvantage of the process is that a denaturing process is necessary after the formation of cDNA
which, in addition to a time delay, also leads to the destruction of the enzyme used to form the cDNA (e.g. thermal denatul~ng). Consequently, additional enzyme must then be added or the sample must be diluted (e.g. addition of reagent), and the sensitivity is reduced.
WO 91/02818 describes a process which, as opposed to EP-A-O 329 822, ismodified in that the 5'-end of the nucleic acid to be amplified hybridizes to the ~-3'-end of the target-specific sequence of the promoter primer. Again, this is a process which requires pretreatment steps in order to obtain a defined ~5'-end of the nucleic acid to be amplified. This pretreatment requires that the nucleic acid to be amplified be purified prior to the actual amplification procedure.
WO 91/02818 andJ. vir. Methods 35, 273-286 (1991), andNature 350 (1991), also describe a process where RNA is amplified under the above listed conditions. In this process, cDNA which corresponds to the analyte RNA is formed in a first step, then the RNA is digested by means of RNAse H, and the treatment of the cDNA is continued as in the detection of analyte DNA.
Because of the substrate specificity of RNAse H, however, this process is limited to RNA analytes only, and many organisms or conditions which are based on characteristic DNA sequences cannot be detected with this process.
Object of the present invention was to find a process for the amplification of nucleic acids which applies to deoxyribonucleic acids and functions on transcription basis and which does not exhibit the above disadvantages known in prior art and is particularly easy to implement.
Subject matter of the invention is, hence, a process for amplifying a segment of a deoxyribonucleic acid A comprising the following steps: - ;
a) converting possibly present double-stranded deoxyribonucleic acid A into single strands resulting in the formation of the strands A- and A+, b) formation of a strand B+, which is at least partially complementary to strand A- by extending a first primer P1+, which contains a nucleotide sequence that is specific for a partial sequence of the segment as well as a promoter-containing nucleotide sequence, --` 2102963 c) formation of a strand B-, which is essentially complementary to strand B+
by extending a second primer P2-, said strands B- and B+ together containing a functional promoter for a DNA-directed RNA polymerase in addition to sequence information from nucleic acid A, d) transcription of the nucleic acid double strand from B- and B+ into ribonucleic acid C+ under control of the promoter, e) formation of a strand D'- which is essentially complementary to C+ by extending a primer, f) formation of a strand D+ which is essentially complementary to D'- by extending a primer and extending D'- to D-, said strands D- and D+
together containing a functional promoter in addition to the sequence information of ribonucleic acid C+, g) transcription of the nucleic acid double strand from D- and D+ into ribonucleic acid C+ while controlling the promoter, while one or several of the reagents for calIying out steps c) to f) are added to the reaction mixture already prior to or simultaneously wi~ the formation of the nucleic acid B+ or with no complete separation be~ween B+ and A- being realized between steps a) and b). ; -Also subject matter of the invention are processes for the detection of deoxyribonucleic acids and reagent kits for the implementation of said processes, particularly for the detection of Listeria monocytogenes.
A deoxyribonucleic acid is understood to be a nucleic acid which is predominantly composed of deoxyribonucleotide components. These deo~yribonucleotides can be of a natural origin. As opposed to natural nucleotides, however, they can also be modified, for example by adding a chemical group to label the deoxyribonucleic acid. In case nucleic acids are used, which are to be extended at ~e 3'-end, these nucleic acids can be blocked at the 5'-end, for example, having a dideoxyribose as deoxyribose. A
segment of a deoxyribonucleic acid as understood in ~e invention can be ~ . .
either a single strand or a double strand. In case of a double-stranded segment,this segment can be double-stranded at both ends (blunt-ended).
A llucleic acid stralld or sequell~c is esselll;ially co~ lel~e~llary to allolher shan~l or sequence, if its nucleo~ide seque~-ce is so complemellt~uy to ~le otllel sequence alat ~e slrands can h yblidize to eacll o~er ove~ tlle wllole sequence of lhis straud. I~is is especially the ~ase, if the strand is produced by a polymerase using the otller st~ d as a te~nplate for the synthesis oftt~is stratld. Ill t}us case the strands may only comprise not complemellta~ ucleotide bases inco1poraled by e~or of ~le polymeraSe.
ucleic acid s~arld or sequence is considered to be at least partially complementa~ to atlother strand o~ sequence, if at Ieast ~ part of ~e strand ca~hybridize to ~e od~er str~nd, the ~1r~d therefore can b~ essentia~ly co~plementaFy o~rer ~ its sequence or can com~se a sequence part which is essentially com~lementary and a sequence pa~t which is not complementaly to ~e o~er s~and.
These pa~ts preferably are more thall ~bouL 10 nucleohdes ~n l~ng~, A sequence is considered to be homologous to anotller sequence, if it hy~idizes to t~e same sequence ~ e othe~ seque~e. Preferably ~ore ~a~ ~0% of ~e U~ U~ vr~ uqll-;q~uqi~ lIU11~iullt;~u~ u~ u~
other soquel~ce. The sequellce C~UI be lollger or sholtel than the odler sequence.
~' ~
~- ~
~ :
... , , .. ... .. . . .. .... I . ~ , , --. 210~3 - 4a-Provided it is hybridized to a deoxylibonucleic acid, a primer is a nucleic acidwhich can act as the starting point for the DNA polymerase activity of an enzyme. For this purpose, tne 3'-end of the primer, especially the nucleotide atthe 3'-end is complementary to the deoxyribonucleic acid and has a free, non-phosphorylated 3'-hydroxyl group. A primer contains a deoxyribonucleotide sequence which is specific for the deoxyribonucleic acid and can hybridize with the segment of the DNA because of its complementarity. This sequence is hereinafter referred to as a target-specific sequence. The location where the primers are supposed to hybridize to the deoxyribonucleic acid can be determined in that the 3'-ends of the primer, with respect to the sequence afterhybridization to the strands, are facing one another. The distance between the 3'-ends of the primers covers a nucleotide sequence of at least one nucleotide, preferably at least 30, more preferably 40-1000 nucleotides. Further, it is alsopreferred that the sequences are not complementary to each other or cannot hybridize to each other. The target-specific sequence has a minimum length of 10 nucleotides, preferably 15-50, a particularly preferred length covers 18-30 nucleotides. Further, the primers are subject to the conditions listed in US-A-4,683,202. The tar~et-specific sequences of the plimers can, however, also be identical or contain identical sequences. They should, however, not be capable of hybridizing to each other.
A promoter-containing nucleotide sequence is a nucleotide sequence which, if it were double-stranded, would initiate the transcription of the nucleic acid ~ ~-segment, following the sequence in 3'-direction by means of an RNA
polymerase. Preferably, the sequence has a single-stranded primer. In a preferred manner, it has a length of 17-100 bases, particularly preferred is a length of 17-50 bases. Suitable double-stranded sequences capable of binding an RNA polymerase are, for example, known from NucleicAcids Research 12, pages 7035-7056 (1984) and Pro~ein Sequences and DNA Analysis 1, pages 269-280 ~1988J, Biophysical Chemis~ry, Part III, pages I I83-1211, Freeman & Co., San Francisco, 1980; J. Bacteriol 170, pages 5248-5256 (1988J; Biochem. ~ 224, pages 799-815 ~1984); Gene Acal. Tech 6, pages 2 9 6 ~
29-32 (1989), EP-A-O 292 S02 and Nucleic Acid Probes, ed. Symons (C~C
Press, Boca Raton, 1989).
The conditions under which primer extension reactions occur are known to the expert from text books, but are also described in US-A-4,683,202. Under the given conditions, the result is an extension product of a primer which can serve as a template for the extension of another primer, and so forth.
In the invention, the term partially complementary means sequences which are either completely complementaty to another nucleic acid or to a part of another nucleic acid or complementaly to another nucleic acid or part of another nucleic acid to such an extent that they can hybridize with the other nucleic acid under the conditions of the extension reaction.
A fimctional promoter is a double-stranded nucleic acid segment which starts the target-specific synthesis of RNA by recognizing and binding RNA
polymerase.
DNA-directed RNA polymerases are known to the expert. They operate promoter-dependent. Examples include the polymerases from phages T7, SP6, N4, or T3. For the transcription system of T7, please refer to Nucleic Acids Research 15, 8783-8798. Transcription is understood to be a process, wherein a ribonucleic acid is formed with ~e aid of a promoter-containing DNA
double strand as a template, said ribonucleic acid being complementary to a -part of a strand of the double strand which is located in transcription direction beginning at the promoter-containing sequence of the plimer. ~ a particularly preferred manner of the invention, the ribonucleic acid formed is complernentaIy to the strand of the DNA, which is the basis for the ;~
amplification procedure of the invention. -RNAse H is an enzyme which digests the RNA strand of an RNA/DNA ~ ~ ~
hybrid, but leaves single-stranded RNA essentially undigested. It is also ~ ~ -possible to use other enzymes, provided they catalyze isothermal strand - -separation.
.., }.''..'-;,.,.,.,.';~' ~` 21029~3 A label as understood in the present invention is a directly or indirectly detectable group L. Directly detectable groups are, for example, radioactive (32p), colored, or fluorescent groups or metal atoms. Indirectly detectable groups include, for example, immunologically or enzymatically active compounds such as antibodies, antigens, haptens, or enzymes or enzymatically active partial enzymes. They are detected in a subsequent reaction or reaction sequence. Particularly preferred are haptens as nucleoside triphosphates (rNTP
or dNTP) labeled with haptens are particularly suitable as substrates of (RNA
or DNA) polymerases. Further it is simple to join a reaction with the labeled antibody to the hapten or the haptenized nucleoside. Such nucleoside triphosphates include, for example, bromide nucleoside triphosphate or digoxigenin, digoxin or fluorescein-coupled nucleoside triphosphates. The steroids listed in EP-A-O 324 474 and their detection have proven to be particularly suitable. For their incorporation in the nucleic acids, please refer to EP-A-O 324 474.
The method of the invention is a special embodiment of so-called hybridization tests whose fundamentals are known to the expert in the field of nucleic acid diagnostics. For experimental details which are not listed hereinafter, please refer to the following publications: "Nucleic acid hybridization", B.D. Hames and S.J. Higgins (edts.), IRL Press, 1986, ~ -~
especiallychapters 1 (HybridizationStrategy), 3 (QuantitativeAnalysisof Solution Hybridization) and 4 (Quantitative Filter Hybridization); "Current Protocols in Molecular Biology", F.M. Ausubel et al. (edt.), J. Wiley and Son, 1987, especially 2.9.1 - 2.9.10, and "Molecular Cloning", J. Sambrook et al. -(edt.), CSH, 1989, especially 9.4.7 - 9.5.8. These experimental details include in particular known processes for the preparation of labeled nucleoside triphosphates as described in EP-A-0 324 474, the chemical synthesis of modified and nonmodified oligonucleotides; further cleaving nucleic acids by ~ -means of restriction enzymes, selecting the hybridization conditions to achieve a certain specificity which depends on the homology between the nucleic acids to be hybridized, their GC contents and ~eir lengths, and include the formation of nucleic acids from nucleoside triphosphates by means of polymerases, if necessary with the aid of pnmers.
Processes for the amplification of nucleic acids serve the purpose of increasingthe number of copies of a given nucleic acid. Object of the process of the invention is the amplification of a segment of a deoxylibonucleic acid. In particular, this segment is not located at one of the two ends of the deoxyribonucleic acid. However, it is understood that the present process can be used to amplify several segments of a deoxyribonucleic acid which are separated from each other or located adjacent to each other. The method of the invention allows a great range of variation as regards both the position and length of the segment. A preferred segment has a length of more than 25 nucleotides, particularly preferred is a length between 30 and 1000 nucleotides.
A process for amplifying nucleic acids as understood by the invention includes ~ ~ ~
also a process where the sequence information of a segment of a nucleic acid - -A is amplified. Sequence information refers to the type and sequence of nucleotide bases at the sugar phosphate residue of the nucleic acid. The method of the invention is sequence-specific, i.e. it is possible to amplify only certain sequences or nucleic acids by selecting the primer sequences and to leaveotheravailablesequenceslargelyunamplified.
The deoxyribonucleic acid A to be amplified can be of any desired origin. It ~ -can be isolated from organisms or be present in organisms, but may also be chemically or enzymatically synthesized. Possible organisms include, for example, viruses and bacteria, but also animal, vegetable, or human cells. It is, however, understood that the present process can be used to amplify several segments of a deo~ibonucleic acid which are separated from or adjacent to one another, and both the position and the length of the segment may valy in wide limits. A segment has a preferred length of more than 25 nucleotides, a particularly preferred length is between 30 and 1000 nucleotides. The DNA
can be pretreated in various ways, which may also depend upon a possible isolation from the organism. Extraction, concentration, amplification, restriction, or cDNA formation are possible, but not necessary to implement the steps of the method of dle invention.
2102~63 , ~
The deoxyribonucleic acid A can be present as strand A in a single stranded form or as a double strand consisting of the strands A- and A+. If present as a double strand, it must be converted into a single-stranded form in a procedural step "a". The expert is familiar with the various ways of denaturing in order toachieve this. A preferred manner of conversion is thermal denatunng, i.e.
heating the sample to exceed the melting point of the deoxyribonucleic acid A.
If the sample to be analyzed contains only one strand of the deoxyribonucleic acid A, said strand is subject to the amplification procedure. If both strands of the segment of the deoxyribonucleic acid A to be amplified are present in the sample, at least one of the two strands is amplified. The one strand of the deoxyribonucleic acid A which is ultimately amplified is hereinaftèr referred to as A-. Before the assay is started, a decision must be made as to which one of the two strands of a double-stranded deoxyribonucleic acid A serves as the basis for the method of the invention and is, hence, referred to as A-. Once made, the decision cannot be reversed.
The references - and + are subsequently used to characterize nucleic acid strands, in order to determine their orientation with respect to complementarity. Strand A- is, for example, at least partially complementary to strand A+. The - and + signs are not to be understood as references to a possible translation. ~`
On a molecular basis, step a is followed by the reactions b - g. If these reactions are not stopped, steps e - g subsequently occur again using ~e respectively formed ribonucleic acid C+ one or several times. This requires the addition of the primers Pl+, P2-, a DNA-directed RNA polymerase, ribonucleotides, deoxyribonucleotides, and additives that serve to adjust the conditions for steps b - d or b - g. To accomplish the object of the invention, it is essential to add one or several of the reagents necessary to carry out steps c -f already prior to or simultaneously with the formation of the nucleic acid B+.
These reagents include in particular the second primer P2-, a DNA-directed RNA polymerase and ribonucleoside triphosphates. In processes known from prior art, these reagents are only added after the fonnation of nucleic acid B+
and after denatuling the double strand of A- and B+.
2102~)3 .
In reaction b, a first primer Pl+ is hybridized with strand A- in a region PT1-.For this purpose, the first primer Pl+ has a nucleotide sequence PT1+ at its 3'-end. Said nucleotide sequence PT1+ is essentially complementary to segment PT1- of strand A-, and under the given conditions, it can hybridize with ~is segment. Especially at the 3'-end, the nucleotides are perfectly complementary to the corresponding nucleotides in segment PTl-. In a preferred manner, the 3'-end of the PTl- segment is separated from the 3'-end of strand A- by a nucleotide sequence segment X-. This segment is at least 1, preferably more than 5, and more particularly more than 30 nucleotides long, and the nucleotides of segment Xl- are essentially not complementary to the corresponding nucleotides on primer Pl+. This ensures that the 3'-end of strand A- does not hybridize with primer P1+.
In addition to the nucleotide sequence PTl+, primer Pl+ contains in 5' direction of PTl+ a nucleotide sequence PPl+, which corresponds to a strand of the promoter sequence. This sequence is subsequently referred to as promoter-containing. It turned out that the use of the sense strand of the promoter sequence is preferred with respect to the sensitivity of the reaction.
Moreover, p~imer Pl+ can contain additional nucleotide sequences PXl+ 5' of the sequence PTl+, preferably between sequence PTl+ and sequence PP1+. In a preferred manner, the sequences PXl+ are not complementary to sequences on strand A-. These may be enhancer sequences, for example.
In a preferred manner, primer Pl+ is extended by adding monodeoxyribo-nucleotide units to the 3'-end of primer Pl+. Each of the mononucleotides added is complementary to the corresponding nucleotide of strand A-. The new segment formed by adding nucleotides is hereina~ter refeIred to as T+. Its length depends on the ability of the enzyme used for the extension to catalyze the formation of long nucleic acids. This ability of the enzyme also determines the nucleotide sequence of the second primer P2-, for the sequence PT2- must be selected such that it can hybridize with B+ within the T+ segment. PT2- is a -segment which contains nucleotides that are complementary to the nucleotides of segrnent T+. -21~2'~63 For both primers, a DNA polymerase is the enzyme used to extend the primers. In a preferred manner, the polymerase for both primers is the same. A
particularly preferred enzyrne is reverse transcriptase as it accepts both RNA
as well as DNA single strands as templates.
In addition to the PT2- segment, prirner P2- may contain additional nucleotides or segments, which are preferably linked to the 5'-end of PT2-.
The nucleotides at the 3'-end of PT2- are essentially complementary, preferably exactly complementary to the corresponding nucleotides of the PT2+ segment and correspond to the respective nucleotides of the A- strand.
The 5'-end of PT2- can be located inside or outside the 5'-end of A+.
The second prirner P2- is added to the reaction mixture preferably prior to or simultaneously with the formation of the nucleic acid B+. The former case can be realized by adding P2- together with P l+ to the mixture containing the A-strand. In a preferred manner, there is no complete separation of the B- and A+
strands, i.e. no denaturing, especially no thermal denatDg after the addition of Pl+, and in a particularly preferred manner, there is no denaturing between the addition of P1+ and the extension of a second primer P2-.
Yet, primer P2- is extended to become a strand B- by using the B+ strand as template, with nucleotide units being added to the 3'-end of P2-. This results in a formation of a strand B-, which contains the segments PTl-, PXl-, and PPl-.
Together with the strand B+, strand B- forms a doubled-stranded nucleic acid, which contains a functional promoter for a DNA-undirected RNA polymerase as well as the sequences PT1 and PT2.
This nucleic acid is now transcribed into a ribonucleic acid C+ under the conditions listed in the pnor art publication EP-A-O 329 822. Subsequently, a strand D'- is formed which is essentially complementaIy to strand C+. This D'- strand serves as a template for the formation of a strand D, resulting in the fonnation of a new nucleic acid, which contains a functional promoter and the segrnents PTl and PT2 as does the hybrid consisting of B- and B+. A strand D- is forrned from the interrnediate strand D'- by extending the latter at the 3'-end with the aid of Pl+ as a template. The result is a system in which the number of the nucleic acids C+, D'-, D- and D+ forrned in the reaction 21V2')63 sequence is amplified. Provided a release of the deoxyribonucleic acid D'- is realized by digestion with RNAase H, a system will result which can operate at an almost constant temperature, i.e. no operation in temperature cycles. In apreferred case, the enzyme for step e) is the same as the one used for step b).
A particularly preferred enzyme is reverse transcriptase.
An advantage of the method of the invention is that a complete denaturing of deoxyribonucleic acid double strands is not necessary after the single-stranded nucleic acid A- is obtained. This means less operational steps and saving instruments to implement the temperature increase/decrease, or saving at least one reagent addition step when chemicals are used for the denaturing procedure. Note that in the detection of nucleic acids as they are present in samples in only minute amounts, it is crucial to avoid contamioation. For this reason, it is desirable to open the reaction vessel as rarely as possible. In the present case, all reagents necessary for the amplification can be added to the ~ ;
prepared sample mixture together with the single-stranded nucleic acid A-.
Then, the reagent vessel must not be opened again until the resulting nucleic acid products C+, D-, or D+ are either removed or taken out for further processing. Moreover, it is an advantage of the method of the invention that - ;
only very small reagent volumes are added when all reagents are added at once such that the sample liquid is diluted to a much smaller extent than in prior art ~ -processes. This renders the method of the invention much more sensitive as it were the case with sequential addition of reagent and intermediate denaturing.
Another advantage of the method of the invention is the use of nucleic acids -A- witnout requiring pretreatment by means of restriction enzymes. This also ; -allows the amplification of the segments of genomic DNA in a simple maoner.
Note that since the restriction step is no longer necessary, the following purification of the nucleic acids to remove possibly present restriction enzymes is no longer necessary.
Yet another advantage is that there is no time delay waiting for the formation of cDNA from the analyte DNA to occur which brings about considerable process advantages even when automated systems are used. -2I 0~963 The simultaneous addition of all reagents necessary for the amplification includes advantages with respect to the final volume of the amplification mixture (dilution effect, smaller volurnes are possible) and for the handling (pipetting errors). Moreover, it is advantageous to add the primers Pl+ and P2-in equal amounts. This can be accomplished in a particularly simple and reproducible manner in one single addition step. The method of the invention is particularly suited for the amplification and the detection of parts of chromosomal DNA.
Analogous to US-A-4 683 202, the method of the invention can also be used in connection with the variant where so-called "nested" primers are employed. In ~
this case, the preamplification according to the invention is followed by the ~ -use of one or two primers whose target-specific sequences are within the amplified segment. This increases the selectivity of the amplification.
Another subject matter of the invention is a method for the detection of deoxyribonucleic acids A by completing steps a to g (if desired steps e-g several times) of the method according to claim 1 and detection of the formed nucleic acid C+, D'-, D-, or D+ or hybrids thereo The implementation of the method according to clairn 1 leads to an increased formation of nucleic acids C+~ D'-, D-, D+, and hybrids thereof. The method of the invention for the detection of nucleic acids makes use of at least one of the so-forrned formed nucleic acids to deterrnine the presence or the amount of deoxyribonucleic acid A in the sample. While caIIying out steps a - g as indicated in claim 1, the detection of the ~ormed nucleic acids C+, D'-, D-, D+, or hybrids thereof can be carried out in a known manner. In ~is case, one or several of the reagents for calIying out steps c - f are added to the reaction mixture already prior to or simultaneously with the formation of nucleic acid B+, or there is no complete separation of B- from A+ between steps a and b. Such known steps include, for example, the hybridization of the above-listed nucleic acids with a labeled nucleic acid probe and detec~ng ~e ~ormed hybrid, e.g. according to US-A-4,58 1,333 or EP-A-0 079 139. When detecting the hybrids of the nucleic acids C+, D- and D+, these nucleic acids must be converted into single strands prior to hybridizing with a single-stranded probe.
~ 2102g63 In a particularly preferred embodiment of the method of the invention, step d) includes the incorporation of a label into the ribonucleic acid C+ by means of a labeled ribonucleoside triphosphate. The label can be either a detectable or an immobilizable group. In this case, the formed nucleic acid C can be detected either directly or after immobilization to a solid phase.
Also subject matter of the invention are reagent kits for the above-listed methods. These kits contain - a description of a method according to claim l; -- a container 1 with an enzyme mixb~re containing an RNA polymerase, a container 2 containing two primers with opposite senses, one of which contains at least one promoter sequence, and containing an enzyme with -reverse transcriptase activity; further, a container 3 containing deo~ibonucleotides and ribonucleotides as well as the necessary additives in one or several suitable containers while the contents of one of several containers may be combined in one;
- a packaging.
Moreover, a reagent kit for the detection of deoxyribonucleic acid A
preferably comes with a container with a labeled nucleic acid probe S.
Yet, another subject matter of the invention is a set o oligonucleotides for the detection of bacteria of the ~amily Listeria containing at least two Listeria-specific primers 1 and 2, which essentially do not hybridize with themselves under the conditions applying to the hybridization of oligonucleotides with the nucleic acids from Listeria. The primers are such that the extension product of the one primer can serve as a template for the extension of the second primer.
Such a set may, for example, consi~t of oligonucleotides which contain nucleotide sequences that are to at least 90 /0, preferably 95 % homologous to ~e Listeria-specif;c sequences of the SEQ ID NO 1-2 and 1-3 and have a length between 10 and 30 nucleotides, preferably between 15-25 nucleotides.
In a particularly preferred manner, the oligonucleotides have an identical l4 ~2~63 length or are longer than the Listeria-specific segments of the oligonucleotidesof the SEQ ID NO. 1-2 or 1-3.
The preferred distance of the 3'-ends of the primer facing each other with respect to the position of the corresponding nucleotides on the Listeria genome are 20 to 500, particularly preferred is a distance of 100 to 350 nucleotides.
One or several primers (preferably at their 5'-ends), as well as the probe may - -have additional nucleotides which are not Listeria-specific. Such nucleotides may serve in the detection of the extension products. Suitable nucleotides are, for example, promoter sequences (particularly for the primers in case of a transcription amplification according to the method of the invention), or nucleotide sequences which can hybridize to other, non-Listeria-specific nucleic acids for the purpose of detection or immobilization.
Under tne hybridization conditions of the primer elongation, the primers are specific for Listeria or preferably a species of Listeria (preferably L. monocytogenes). In a preferred manner, the probe is rendered specific for Listeria monocytogenes by using a DNA polymerase. In a particularly preferred manner, the probe carries a label which allows immobilization to occur.
The positions of the Listeria monocytogenes DNA to which the Listeria-specific segments of the primers hybridize also allows another definition of theListeria primers. The following data refers to the sequence of the plasmid pPLM 63 of WO 90/08197.
; . . .. . _ .
Position _ ._ Primer 1 35 - 56 Primer 2 196 - 216 Probe S 162 - 180 Pl+ is the preferred primer 1, P2- is the preferred primer 2, and NAS 4 is the preferred probe.
21 0~.g63 The set of oligonucleotides of the invention allows a partisularly specific amplification/detection of Listeria DN~. A method, primer, or probe is referred to as specific, if detection of Listeria, especially Listeria monocytogenes is possible without detecting nucleic acids from other microorganisms which may occur in the sample to be examined.
Figure 1 is a representation of the reference numerals used in the description.
Figure la) is a diagrammatic representation of a hybrid of A- and Pl+ as it will form after adding Pl+ to A- to generate an extension of Pl+ (arrow).
Figure lb) is a diagrammatic representation of the s~ucture of primer Pl+.
Figure lc) is a diagrammatic representation of the structure of B+, where the extension direction of P2- is also indicated.
Figure ld) is a diagrammatic representation of the structure of B-.
Figure le) is a diagramrnatic representation of the structure of C+.
Figure lf) is a diagrammatic representation of the structure of D'-.
Figure lg) is a diagrammatic representation of the structure of the hybrids of D+ and D-.
Figure 2 is a diagranunatic representation of a particularly preferred variant of the method of the invention where all the reagents are added after the denaturing of A.
Figure 3 shows a variant of the method of the invention where all the reagents are added prior to step b), except for those necessary for the transcription. ~-Figure 4 shows a variant where only NTPs and, if desired, RNAse H are added after step b.
Figure S shows a variant where Pl+, P2- and nucleoside triphosphates are added prior to or during the denaturing process of the DNA A, but where the enzymes are added after step a, but prior to step b. -;
~ 2102963 Figure 6 is a diagrammatic representation of the PIII/dthl4/dthl8 region of the Listeria monocytogenes chromosomes and also shows the position of the primer Pl+ (SEQ ID NO 1), P2- (SEQ ~D NO 2), as well as the capture probe S (NAS 4) (SEQ ID NO 3) (all specific for L. monocytogenes).
Figure 7 shows an autoradiogram of the amplification products where the products are separated according to size by agarose gel electrophoresis, then blotted onto a nylon membrane and detected with a digoxigenin-labeled oligonucleotide probe. The amplified ribonucleic acid C+ runs as a product with a length of 181 nt. M refers to the molecule weight label. Lanes 1-9 carry different amounts of chromosomal Listeria DNA (lane 1 50 ng, lane 2 5 ng, lane 3 500 pg, lane 4 50 pg, lane 5 5 pg, lane 6 500 fg, lane 7 50 fg, lane 8 5 fg, lane 9 no chromosomal Listeria DNA).
The following examples explain the invention in greater detail:
Example 1 Oligonucleotides The oligonucleotides of the sequences ID NO 1, 2, and 3 were produced with the aid of a DNA synthesizer (Applied Biosystems) from unmodified and/or digoxigenated mononucleotides (EP-A-O 324 474).
Denaturing chromosomal DNA
The denaturing reaction was allowed to occur in a volume of 16 ,~IL The reaction mixture was composed as follows:
62.5 mM Tris-HCI, pH 8.5;
78.12S mM KCI;
17.75 mM MgC12;
1.5625 mM per dNTP (Pharmacia);
3.125 mM per NTP (Pharmacia) and 50 ng to 5 fg purified chromosomal DNA
of Listeria monocytogenes.
2102')63 The reaction mixture was allowed to boil for 15 min and then immediately cooled on ice.
Amplifving denatured chromosomal DNA
The amplification reaction was allowed to occur in a volume of 25 ~11 in an Eppendorf vessel. For this reaction to occur, 9 1ll of a second mixture, consisting of DTT, DMSO, primer Pl+ and Pl-, RNasin, RNase H, T7 RNA
polymerase, AMV reverse transcriptase, and BSA (all enzymes and BSA by Boehringer Mannheim) was added to the denaturing tlux such that the final concentration of the amplification mixture was as follows:
40 rnM Tris-HCI, pH 8.5;
50mMKCI;
12 mM MgClz;
10 mM DTT;
15 % DMSO;
0.2 ',lM primer Pl+;
0.2 ~,lM prirner Pl-;
1 mM per dNTP;
2 mM per NTP;
12.5 URNasin;
80 U T7 RNA polymerase;
Samples, such as body fluids, contain deoxyribonucleic acids only in minute amounts. These contain, however, sequence information which is often a fairly reliable indicator for the presence of certain organisms or the conditions of such organisms. Amplifying the sequence info~nation has proven to be a usefill tool. While the first applicable amplification systems were based on ~e exponential amplification of sequence information in temperature cycles, more recent systems allow these reactions to occur at almost constant temperatures.
Such a system is described in EP-A-O 329 822, for example. This publication describes a process for amplifying ribonucleic acids where an antisense-p~omoter-containing oligonucleotide is hybridized to the one end of the nucleic acid to be amplified. Using the nucleic acid as a template, this oligonucleotide is extended by adding deoxyTibonucleotides. After the RNA/DNA hybrid is denatured, another oligonucleotide is hybridized to the end of the DNA which is remote from the promoter sequence and is extended under the formation of a functional promoter.
The segment of the double strand which follows the promoter is then transcribed into RNA under control of the promoter. Then, the RNA is again used to synthesize the promoter-containing DNA double strand. A great disadvantage of this process is that if DNA is used as the initial template, thecorresponding sequence is either chemically or synthetically produced or cleaved out of a plasmid by means of a restriction enzyme such ~at the nucleotides of the nucleic acid to be amplified which are located at the 5'-end hybridi~e to the promoter oligonucleotide. Yet another disadvantage of the process is that a denaturing process is necessary after the formation of cDNA
which, in addition to a time delay, also leads to the destruction of the enzyme used to form the cDNA (e.g. thermal denatul~ng). Consequently, additional enzyme must then be added or the sample must be diluted (e.g. addition of reagent), and the sensitivity is reduced.
WO 91/02818 describes a process which, as opposed to EP-A-O 329 822, ismodified in that the 5'-end of the nucleic acid to be amplified hybridizes to the ~-3'-end of the target-specific sequence of the promoter primer. Again, this is a process which requires pretreatment steps in order to obtain a defined ~5'-end of the nucleic acid to be amplified. This pretreatment requires that the nucleic acid to be amplified be purified prior to the actual amplification procedure.
WO 91/02818 andJ. vir. Methods 35, 273-286 (1991), andNature 350 (1991), also describe a process where RNA is amplified under the above listed conditions. In this process, cDNA which corresponds to the analyte RNA is formed in a first step, then the RNA is digested by means of RNAse H, and the treatment of the cDNA is continued as in the detection of analyte DNA.
Because of the substrate specificity of RNAse H, however, this process is limited to RNA analytes only, and many organisms or conditions which are based on characteristic DNA sequences cannot be detected with this process.
Object of the present invention was to find a process for the amplification of nucleic acids which applies to deoxyribonucleic acids and functions on transcription basis and which does not exhibit the above disadvantages known in prior art and is particularly easy to implement.
Subject matter of the invention is, hence, a process for amplifying a segment of a deoxyribonucleic acid A comprising the following steps: - ;
a) converting possibly present double-stranded deoxyribonucleic acid A into single strands resulting in the formation of the strands A- and A+, b) formation of a strand B+, which is at least partially complementary to strand A- by extending a first primer P1+, which contains a nucleotide sequence that is specific for a partial sequence of the segment as well as a promoter-containing nucleotide sequence, --` 2102963 c) formation of a strand B-, which is essentially complementary to strand B+
by extending a second primer P2-, said strands B- and B+ together containing a functional promoter for a DNA-directed RNA polymerase in addition to sequence information from nucleic acid A, d) transcription of the nucleic acid double strand from B- and B+ into ribonucleic acid C+ under control of the promoter, e) formation of a strand D'- which is essentially complementary to C+ by extending a primer, f) formation of a strand D+ which is essentially complementary to D'- by extending a primer and extending D'- to D-, said strands D- and D+
together containing a functional promoter in addition to the sequence information of ribonucleic acid C+, g) transcription of the nucleic acid double strand from D- and D+ into ribonucleic acid C+ while controlling the promoter, while one or several of the reagents for calIying out steps c) to f) are added to the reaction mixture already prior to or simultaneously wi~ the formation of the nucleic acid B+ or with no complete separation be~ween B+ and A- being realized between steps a) and b). ; -Also subject matter of the invention are processes for the detection of deoxyribonucleic acids and reagent kits for the implementation of said processes, particularly for the detection of Listeria monocytogenes.
A deoxyribonucleic acid is understood to be a nucleic acid which is predominantly composed of deoxyribonucleotide components. These deo~yribonucleotides can be of a natural origin. As opposed to natural nucleotides, however, they can also be modified, for example by adding a chemical group to label the deoxyribonucleic acid. In case nucleic acids are used, which are to be extended at ~e 3'-end, these nucleic acids can be blocked at the 5'-end, for example, having a dideoxyribose as deoxyribose. A
segment of a deoxyribonucleic acid as understood in ~e invention can be ~ . .
either a single strand or a double strand. In case of a double-stranded segment,this segment can be double-stranded at both ends (blunt-ended).
A llucleic acid stralld or sequell~c is esselll;ially co~ lel~e~llary to allolher shan~l or sequence, if its nucleo~ide seque~-ce is so complemellt~uy to ~le otllel sequence alat ~e slrands can h yblidize to eacll o~er ove~ tlle wllole sequence of lhis straud. I~is is especially the ~ase, if the strand is produced by a polymerase using the otller st~ d as a te~nplate for the synthesis oftt~is stratld. Ill t}us case the strands may only comprise not complemellta~ ucleotide bases inco1poraled by e~or of ~le polymeraSe.
ucleic acid s~arld or sequence is considered to be at least partially complementa~ to atlother strand o~ sequence, if at Ieast ~ part of ~e strand ca~hybridize to ~e od~er str~nd, the ~1r~d therefore can b~ essentia~ly co~plementaFy o~rer ~ its sequence or can com~se a sequence part which is essentially com~lementary and a sequence pa~t which is not complementaly to ~e o~er s~and.
These pa~ts preferably are more thall ~bouL 10 nucleohdes ~n l~ng~, A sequence is considered to be homologous to anotller sequence, if it hy~idizes to t~e same sequence ~ e othe~ seque~e. Preferably ~ore ~a~ ~0% of ~e U~ U~ vr~ uqll-;q~uqi~ lIU11~iullt;~u~ u~ u~
other soquel~ce. The sequellce C~UI be lollger or sholtel than the odler sequence.
~' ~
~- ~
~ :
... , , .. ... .. . . .. .... I . ~ , , --. 210~3 - 4a-Provided it is hybridized to a deoxylibonucleic acid, a primer is a nucleic acidwhich can act as the starting point for the DNA polymerase activity of an enzyme. For this purpose, tne 3'-end of the primer, especially the nucleotide atthe 3'-end is complementary to the deoxyribonucleic acid and has a free, non-phosphorylated 3'-hydroxyl group. A primer contains a deoxyribonucleotide sequence which is specific for the deoxyribonucleic acid and can hybridize with the segment of the DNA because of its complementarity. This sequence is hereinafter referred to as a target-specific sequence. The location where the primers are supposed to hybridize to the deoxyribonucleic acid can be determined in that the 3'-ends of the primer, with respect to the sequence afterhybridization to the strands, are facing one another. The distance between the 3'-ends of the primers covers a nucleotide sequence of at least one nucleotide, preferably at least 30, more preferably 40-1000 nucleotides. Further, it is alsopreferred that the sequences are not complementary to each other or cannot hybridize to each other. The target-specific sequence has a minimum length of 10 nucleotides, preferably 15-50, a particularly preferred length covers 18-30 nucleotides. Further, the primers are subject to the conditions listed in US-A-4,683,202. The tar~et-specific sequences of the plimers can, however, also be identical or contain identical sequences. They should, however, not be capable of hybridizing to each other.
A promoter-containing nucleotide sequence is a nucleotide sequence which, if it were double-stranded, would initiate the transcription of the nucleic acid ~ ~-segment, following the sequence in 3'-direction by means of an RNA
polymerase. Preferably, the sequence has a single-stranded primer. In a preferred manner, it has a length of 17-100 bases, particularly preferred is a length of 17-50 bases. Suitable double-stranded sequences capable of binding an RNA polymerase are, for example, known from NucleicAcids Research 12, pages 7035-7056 (1984) and Pro~ein Sequences and DNA Analysis 1, pages 269-280 ~1988J, Biophysical Chemis~ry, Part III, pages I I83-1211, Freeman & Co., San Francisco, 1980; J. Bacteriol 170, pages 5248-5256 (1988J; Biochem. ~ 224, pages 799-815 ~1984); Gene Acal. Tech 6, pages 2 9 6 ~
29-32 (1989), EP-A-O 292 S02 and Nucleic Acid Probes, ed. Symons (C~C
Press, Boca Raton, 1989).
The conditions under which primer extension reactions occur are known to the expert from text books, but are also described in US-A-4,683,202. Under the given conditions, the result is an extension product of a primer which can serve as a template for the extension of another primer, and so forth.
In the invention, the term partially complementary means sequences which are either completely complementaty to another nucleic acid or to a part of another nucleic acid or complementaly to another nucleic acid or part of another nucleic acid to such an extent that they can hybridize with the other nucleic acid under the conditions of the extension reaction.
A fimctional promoter is a double-stranded nucleic acid segment which starts the target-specific synthesis of RNA by recognizing and binding RNA
polymerase.
DNA-directed RNA polymerases are known to the expert. They operate promoter-dependent. Examples include the polymerases from phages T7, SP6, N4, or T3. For the transcription system of T7, please refer to Nucleic Acids Research 15, 8783-8798. Transcription is understood to be a process, wherein a ribonucleic acid is formed with ~e aid of a promoter-containing DNA
double strand as a template, said ribonucleic acid being complementary to a -part of a strand of the double strand which is located in transcription direction beginning at the promoter-containing sequence of the plimer. ~ a particularly preferred manner of the invention, the ribonucleic acid formed is complernentaIy to the strand of the DNA, which is the basis for the ;~
amplification procedure of the invention. -RNAse H is an enzyme which digests the RNA strand of an RNA/DNA ~ ~ ~
hybrid, but leaves single-stranded RNA essentially undigested. It is also ~ ~ -possible to use other enzymes, provided they catalyze isothermal strand - -separation.
.., }.''..'-;,.,.,.,.';~' ~` 21029~3 A label as understood in the present invention is a directly or indirectly detectable group L. Directly detectable groups are, for example, radioactive (32p), colored, or fluorescent groups or metal atoms. Indirectly detectable groups include, for example, immunologically or enzymatically active compounds such as antibodies, antigens, haptens, or enzymes or enzymatically active partial enzymes. They are detected in a subsequent reaction or reaction sequence. Particularly preferred are haptens as nucleoside triphosphates (rNTP
or dNTP) labeled with haptens are particularly suitable as substrates of (RNA
or DNA) polymerases. Further it is simple to join a reaction with the labeled antibody to the hapten or the haptenized nucleoside. Such nucleoside triphosphates include, for example, bromide nucleoside triphosphate or digoxigenin, digoxin or fluorescein-coupled nucleoside triphosphates. The steroids listed in EP-A-O 324 474 and their detection have proven to be particularly suitable. For their incorporation in the nucleic acids, please refer to EP-A-O 324 474.
The method of the invention is a special embodiment of so-called hybridization tests whose fundamentals are known to the expert in the field of nucleic acid diagnostics. For experimental details which are not listed hereinafter, please refer to the following publications: "Nucleic acid hybridization", B.D. Hames and S.J. Higgins (edts.), IRL Press, 1986, ~ -~
especiallychapters 1 (HybridizationStrategy), 3 (QuantitativeAnalysisof Solution Hybridization) and 4 (Quantitative Filter Hybridization); "Current Protocols in Molecular Biology", F.M. Ausubel et al. (edt.), J. Wiley and Son, 1987, especially 2.9.1 - 2.9.10, and "Molecular Cloning", J. Sambrook et al. -(edt.), CSH, 1989, especially 9.4.7 - 9.5.8. These experimental details include in particular known processes for the preparation of labeled nucleoside triphosphates as described in EP-A-0 324 474, the chemical synthesis of modified and nonmodified oligonucleotides; further cleaving nucleic acids by ~ -means of restriction enzymes, selecting the hybridization conditions to achieve a certain specificity which depends on the homology between the nucleic acids to be hybridized, their GC contents and ~eir lengths, and include the formation of nucleic acids from nucleoside triphosphates by means of polymerases, if necessary with the aid of pnmers.
Processes for the amplification of nucleic acids serve the purpose of increasingthe number of copies of a given nucleic acid. Object of the process of the invention is the amplification of a segment of a deoxylibonucleic acid. In particular, this segment is not located at one of the two ends of the deoxyribonucleic acid. However, it is understood that the present process can be used to amplify several segments of a deoxyribonucleic acid which are separated from each other or located adjacent to each other. The method of the invention allows a great range of variation as regards both the position and length of the segment. A preferred segment has a length of more than 25 nucleotides, particularly preferred is a length between 30 and 1000 nucleotides.
A process for amplifying nucleic acids as understood by the invention includes ~ ~ ~
also a process where the sequence information of a segment of a nucleic acid - -A is amplified. Sequence information refers to the type and sequence of nucleotide bases at the sugar phosphate residue of the nucleic acid. The method of the invention is sequence-specific, i.e. it is possible to amplify only certain sequences or nucleic acids by selecting the primer sequences and to leaveotheravailablesequenceslargelyunamplified.
The deoxyribonucleic acid A to be amplified can be of any desired origin. It ~ -can be isolated from organisms or be present in organisms, but may also be chemically or enzymatically synthesized. Possible organisms include, for example, viruses and bacteria, but also animal, vegetable, or human cells. It is, however, understood that the present process can be used to amplify several segments of a deo~ibonucleic acid which are separated from or adjacent to one another, and both the position and the length of the segment may valy in wide limits. A segment has a preferred length of more than 25 nucleotides, a particularly preferred length is between 30 and 1000 nucleotides. The DNA
can be pretreated in various ways, which may also depend upon a possible isolation from the organism. Extraction, concentration, amplification, restriction, or cDNA formation are possible, but not necessary to implement the steps of the method of dle invention.
2102~63 , ~
The deoxyribonucleic acid A can be present as strand A in a single stranded form or as a double strand consisting of the strands A- and A+. If present as a double strand, it must be converted into a single-stranded form in a procedural step "a". The expert is familiar with the various ways of denaturing in order toachieve this. A preferred manner of conversion is thermal denatunng, i.e.
heating the sample to exceed the melting point of the deoxyribonucleic acid A.
If the sample to be analyzed contains only one strand of the deoxyribonucleic acid A, said strand is subject to the amplification procedure. If both strands of the segment of the deoxyribonucleic acid A to be amplified are present in the sample, at least one of the two strands is amplified. The one strand of the deoxyribonucleic acid A which is ultimately amplified is hereinaftèr referred to as A-. Before the assay is started, a decision must be made as to which one of the two strands of a double-stranded deoxyribonucleic acid A serves as the basis for the method of the invention and is, hence, referred to as A-. Once made, the decision cannot be reversed.
The references - and + are subsequently used to characterize nucleic acid strands, in order to determine their orientation with respect to complementarity. Strand A- is, for example, at least partially complementary to strand A+. The - and + signs are not to be understood as references to a possible translation. ~`
On a molecular basis, step a is followed by the reactions b - g. If these reactions are not stopped, steps e - g subsequently occur again using ~e respectively formed ribonucleic acid C+ one or several times. This requires the addition of the primers Pl+, P2-, a DNA-directed RNA polymerase, ribonucleotides, deoxyribonucleotides, and additives that serve to adjust the conditions for steps b - d or b - g. To accomplish the object of the invention, it is essential to add one or several of the reagents necessary to carry out steps c -f already prior to or simultaneously with the formation of the nucleic acid B+.
These reagents include in particular the second primer P2-, a DNA-directed RNA polymerase and ribonucleoside triphosphates. In processes known from prior art, these reagents are only added after the fonnation of nucleic acid B+
and after denatuling the double strand of A- and B+.
2102~)3 .
In reaction b, a first primer Pl+ is hybridized with strand A- in a region PT1-.For this purpose, the first primer Pl+ has a nucleotide sequence PT1+ at its 3'-end. Said nucleotide sequence PT1+ is essentially complementary to segment PT1- of strand A-, and under the given conditions, it can hybridize with ~is segment. Especially at the 3'-end, the nucleotides are perfectly complementary to the corresponding nucleotides in segment PTl-. In a preferred manner, the 3'-end of the PTl- segment is separated from the 3'-end of strand A- by a nucleotide sequence segment X-. This segment is at least 1, preferably more than 5, and more particularly more than 30 nucleotides long, and the nucleotides of segment Xl- are essentially not complementary to the corresponding nucleotides on primer Pl+. This ensures that the 3'-end of strand A- does not hybridize with primer P1+.
In addition to the nucleotide sequence PTl+, primer Pl+ contains in 5' direction of PTl+ a nucleotide sequence PPl+, which corresponds to a strand of the promoter sequence. This sequence is subsequently referred to as promoter-containing. It turned out that the use of the sense strand of the promoter sequence is preferred with respect to the sensitivity of the reaction.
Moreover, p~imer Pl+ can contain additional nucleotide sequences PXl+ 5' of the sequence PTl+, preferably between sequence PTl+ and sequence PP1+. In a preferred manner, the sequences PXl+ are not complementary to sequences on strand A-. These may be enhancer sequences, for example.
In a preferred manner, primer Pl+ is extended by adding monodeoxyribo-nucleotide units to the 3'-end of primer Pl+. Each of the mononucleotides added is complementary to the corresponding nucleotide of strand A-. The new segment formed by adding nucleotides is hereina~ter refeIred to as T+. Its length depends on the ability of the enzyme used for the extension to catalyze the formation of long nucleic acids. This ability of the enzyme also determines the nucleotide sequence of the second primer P2-, for the sequence PT2- must be selected such that it can hybridize with B+ within the T+ segment. PT2- is a -segment which contains nucleotides that are complementary to the nucleotides of segrnent T+. -21~2'~63 For both primers, a DNA polymerase is the enzyme used to extend the primers. In a preferred manner, the polymerase for both primers is the same. A
particularly preferred enzyrne is reverse transcriptase as it accepts both RNA
as well as DNA single strands as templates.
In addition to the PT2- segment, prirner P2- may contain additional nucleotides or segments, which are preferably linked to the 5'-end of PT2-.
The nucleotides at the 3'-end of PT2- are essentially complementary, preferably exactly complementary to the corresponding nucleotides of the PT2+ segment and correspond to the respective nucleotides of the A- strand.
The 5'-end of PT2- can be located inside or outside the 5'-end of A+.
The second prirner P2- is added to the reaction mixture preferably prior to or simultaneously with the formation of the nucleic acid B+. The former case can be realized by adding P2- together with P l+ to the mixture containing the A-strand. In a preferred manner, there is no complete separation of the B- and A+
strands, i.e. no denaturing, especially no thermal denatDg after the addition of Pl+, and in a particularly preferred manner, there is no denaturing between the addition of P1+ and the extension of a second primer P2-.
Yet, primer P2- is extended to become a strand B- by using the B+ strand as template, with nucleotide units being added to the 3'-end of P2-. This results in a formation of a strand B-, which contains the segments PTl-, PXl-, and PPl-.
Together with the strand B+, strand B- forms a doubled-stranded nucleic acid, which contains a functional promoter for a DNA-undirected RNA polymerase as well as the sequences PT1 and PT2.
This nucleic acid is now transcribed into a ribonucleic acid C+ under the conditions listed in the pnor art publication EP-A-O 329 822. Subsequently, a strand D'- is formed which is essentially complementaIy to strand C+. This D'- strand serves as a template for the formation of a strand D, resulting in the fonnation of a new nucleic acid, which contains a functional promoter and the segrnents PTl and PT2 as does the hybrid consisting of B- and B+. A strand D- is forrned from the interrnediate strand D'- by extending the latter at the 3'-end with the aid of Pl+ as a template. The result is a system in which the number of the nucleic acids C+, D'-, D- and D+ forrned in the reaction 21V2')63 sequence is amplified. Provided a release of the deoxyribonucleic acid D'- is realized by digestion with RNAase H, a system will result which can operate at an almost constant temperature, i.e. no operation in temperature cycles. In apreferred case, the enzyme for step e) is the same as the one used for step b).
A particularly preferred enzyme is reverse transcriptase.
An advantage of the method of the invention is that a complete denaturing of deoxyribonucleic acid double strands is not necessary after the single-stranded nucleic acid A- is obtained. This means less operational steps and saving instruments to implement the temperature increase/decrease, or saving at least one reagent addition step when chemicals are used for the denaturing procedure. Note that in the detection of nucleic acids as they are present in samples in only minute amounts, it is crucial to avoid contamioation. For this reason, it is desirable to open the reaction vessel as rarely as possible. In the present case, all reagents necessary for the amplification can be added to the ~ ;
prepared sample mixture together with the single-stranded nucleic acid A-.
Then, the reagent vessel must not be opened again until the resulting nucleic acid products C+, D-, or D+ are either removed or taken out for further processing. Moreover, it is an advantage of the method of the invention that - ;
only very small reagent volumes are added when all reagents are added at once such that the sample liquid is diluted to a much smaller extent than in prior art ~ -processes. This renders the method of the invention much more sensitive as it were the case with sequential addition of reagent and intermediate denaturing.
Another advantage of the method of the invention is the use of nucleic acids -A- witnout requiring pretreatment by means of restriction enzymes. This also ; -allows the amplification of the segments of genomic DNA in a simple maoner.
Note that since the restriction step is no longer necessary, the following purification of the nucleic acids to remove possibly present restriction enzymes is no longer necessary.
Yet another advantage is that there is no time delay waiting for the formation of cDNA from the analyte DNA to occur which brings about considerable process advantages even when automated systems are used. -2I 0~963 The simultaneous addition of all reagents necessary for the amplification includes advantages with respect to the final volume of the amplification mixture (dilution effect, smaller volurnes are possible) and for the handling (pipetting errors). Moreover, it is advantageous to add the primers Pl+ and P2-in equal amounts. This can be accomplished in a particularly simple and reproducible manner in one single addition step. The method of the invention is particularly suited for the amplification and the detection of parts of chromosomal DNA.
Analogous to US-A-4 683 202, the method of the invention can also be used in connection with the variant where so-called "nested" primers are employed. In ~
this case, the preamplification according to the invention is followed by the ~ -use of one or two primers whose target-specific sequences are within the amplified segment. This increases the selectivity of the amplification.
Another subject matter of the invention is a method for the detection of deoxyribonucleic acids A by completing steps a to g (if desired steps e-g several times) of the method according to claim 1 and detection of the formed nucleic acid C+, D'-, D-, or D+ or hybrids thereo The implementation of the method according to clairn 1 leads to an increased formation of nucleic acids C+~ D'-, D-, D+, and hybrids thereof. The method of the invention for the detection of nucleic acids makes use of at least one of the so-forrned formed nucleic acids to deterrnine the presence or the amount of deoxyribonucleic acid A in the sample. While caIIying out steps a - g as indicated in claim 1, the detection of the ~ormed nucleic acids C+, D'-, D-, D+, or hybrids thereof can be carried out in a known manner. In ~is case, one or several of the reagents for calIying out steps c - f are added to the reaction mixture already prior to or simultaneously with the formation of nucleic acid B+, or there is no complete separation of B- from A+ between steps a and b. Such known steps include, for example, the hybridization of the above-listed nucleic acids with a labeled nucleic acid probe and detec~ng ~e ~ormed hybrid, e.g. according to US-A-4,58 1,333 or EP-A-0 079 139. When detecting the hybrids of the nucleic acids C+, D- and D+, these nucleic acids must be converted into single strands prior to hybridizing with a single-stranded probe.
~ 2102g63 In a particularly preferred embodiment of the method of the invention, step d) includes the incorporation of a label into the ribonucleic acid C+ by means of a labeled ribonucleoside triphosphate. The label can be either a detectable or an immobilizable group. In this case, the formed nucleic acid C can be detected either directly or after immobilization to a solid phase.
Also subject matter of the invention are reagent kits for the above-listed methods. These kits contain - a description of a method according to claim l; -- a container 1 with an enzyme mixb~re containing an RNA polymerase, a container 2 containing two primers with opposite senses, one of which contains at least one promoter sequence, and containing an enzyme with -reverse transcriptase activity; further, a container 3 containing deo~ibonucleotides and ribonucleotides as well as the necessary additives in one or several suitable containers while the contents of one of several containers may be combined in one;
- a packaging.
Moreover, a reagent kit for the detection of deoxyribonucleic acid A
preferably comes with a container with a labeled nucleic acid probe S.
Yet, another subject matter of the invention is a set o oligonucleotides for the detection of bacteria of the ~amily Listeria containing at least two Listeria-specific primers 1 and 2, which essentially do not hybridize with themselves under the conditions applying to the hybridization of oligonucleotides with the nucleic acids from Listeria. The primers are such that the extension product of the one primer can serve as a template for the extension of the second primer.
Such a set may, for example, consi~t of oligonucleotides which contain nucleotide sequences that are to at least 90 /0, preferably 95 % homologous to ~e Listeria-specif;c sequences of the SEQ ID NO 1-2 and 1-3 and have a length between 10 and 30 nucleotides, preferably between 15-25 nucleotides.
In a particularly preferred manner, the oligonucleotides have an identical l4 ~2~63 length or are longer than the Listeria-specific segments of the oligonucleotidesof the SEQ ID NO. 1-2 or 1-3.
The preferred distance of the 3'-ends of the primer facing each other with respect to the position of the corresponding nucleotides on the Listeria genome are 20 to 500, particularly preferred is a distance of 100 to 350 nucleotides.
One or several primers (preferably at their 5'-ends), as well as the probe may - -have additional nucleotides which are not Listeria-specific. Such nucleotides may serve in the detection of the extension products. Suitable nucleotides are, for example, promoter sequences (particularly for the primers in case of a transcription amplification according to the method of the invention), or nucleotide sequences which can hybridize to other, non-Listeria-specific nucleic acids for the purpose of detection or immobilization.
Under tne hybridization conditions of the primer elongation, the primers are specific for Listeria or preferably a species of Listeria (preferably L. monocytogenes). In a preferred manner, the probe is rendered specific for Listeria monocytogenes by using a DNA polymerase. In a particularly preferred manner, the probe carries a label which allows immobilization to occur.
The positions of the Listeria monocytogenes DNA to which the Listeria-specific segments of the primers hybridize also allows another definition of theListeria primers. The following data refers to the sequence of the plasmid pPLM 63 of WO 90/08197.
; . . .. . _ .
Position _ ._ Primer 1 35 - 56 Primer 2 196 - 216 Probe S 162 - 180 Pl+ is the preferred primer 1, P2- is the preferred primer 2, and NAS 4 is the preferred probe.
21 0~.g63 The set of oligonucleotides of the invention allows a partisularly specific amplification/detection of Listeria DN~. A method, primer, or probe is referred to as specific, if detection of Listeria, especially Listeria monocytogenes is possible without detecting nucleic acids from other microorganisms which may occur in the sample to be examined.
Figure 1 is a representation of the reference numerals used in the description.
Figure la) is a diagrammatic representation of a hybrid of A- and Pl+ as it will form after adding Pl+ to A- to generate an extension of Pl+ (arrow).
Figure lb) is a diagrammatic representation of the s~ucture of primer Pl+.
Figure lc) is a diagrammatic representation of the structure of B+, where the extension direction of P2- is also indicated.
Figure ld) is a diagrammatic representation of the structure of B-.
Figure le) is a diagramrnatic representation of the structure of C+.
Figure lf) is a diagrammatic representation of the structure of D'-.
Figure lg) is a diagrammatic representation of the structure of the hybrids of D+ and D-.
Figure 2 is a diagranunatic representation of a particularly preferred variant of the method of the invention where all the reagents are added after the denaturing of A.
Figure 3 shows a variant of the method of the invention where all the reagents are added prior to step b), except for those necessary for the transcription. ~-Figure 4 shows a variant where only NTPs and, if desired, RNAse H are added after step b.
Figure S shows a variant where Pl+, P2- and nucleoside triphosphates are added prior to or during the denaturing process of the DNA A, but where the enzymes are added after step a, but prior to step b. -;
~ 2102963 Figure 6 is a diagrammatic representation of the PIII/dthl4/dthl8 region of the Listeria monocytogenes chromosomes and also shows the position of the primer Pl+ (SEQ ID NO 1), P2- (SEQ ~D NO 2), as well as the capture probe S (NAS 4) (SEQ ID NO 3) (all specific for L. monocytogenes).
Figure 7 shows an autoradiogram of the amplification products where the products are separated according to size by agarose gel electrophoresis, then blotted onto a nylon membrane and detected with a digoxigenin-labeled oligonucleotide probe. The amplified ribonucleic acid C+ runs as a product with a length of 181 nt. M refers to the molecule weight label. Lanes 1-9 carry different amounts of chromosomal Listeria DNA (lane 1 50 ng, lane 2 5 ng, lane 3 500 pg, lane 4 50 pg, lane 5 5 pg, lane 6 500 fg, lane 7 50 fg, lane 8 5 fg, lane 9 no chromosomal Listeria DNA).
The following examples explain the invention in greater detail:
Example 1 Oligonucleotides The oligonucleotides of the sequences ID NO 1, 2, and 3 were produced with the aid of a DNA synthesizer (Applied Biosystems) from unmodified and/or digoxigenated mononucleotides (EP-A-O 324 474).
Denaturing chromosomal DNA
The denaturing reaction was allowed to occur in a volume of 16 ,~IL The reaction mixture was composed as follows:
62.5 mM Tris-HCI, pH 8.5;
78.12S mM KCI;
17.75 mM MgC12;
1.5625 mM per dNTP (Pharmacia);
3.125 mM per NTP (Pharmacia) and 50 ng to 5 fg purified chromosomal DNA
of Listeria monocytogenes.
2102')63 The reaction mixture was allowed to boil for 15 min and then immediately cooled on ice.
Amplifving denatured chromosomal DNA
The amplification reaction was allowed to occur in a volume of 25 ~11 in an Eppendorf vessel. For this reaction to occur, 9 1ll of a second mixture, consisting of DTT, DMSO, primer Pl+ and Pl-, RNasin, RNase H, T7 RNA
polymerase, AMV reverse transcriptase, and BSA (all enzymes and BSA by Boehringer Mannheim) was added to the denaturing tlux such that the final concentration of the amplification mixture was as follows:
40 rnM Tris-HCI, pH 8.5;
50mMKCI;
12 mM MgClz;
10 mM DTT;
15 % DMSO;
0.2 ',lM primer Pl+;
0.2 ~,lM prirner Pl-;
1 mM per dNTP;
2 mM per NTP;
12.5 URNasin;
80 U T7 RNA polymerase;
4 U AMV reverse transcriptase; - -1 U RNase H and 0.1 ~,lg/lll BSA.
Then the reaction vessel was clo~ed and not opened again until the amplification procedure was stopped. The reaction was allowed to occur for -~
90 min at 40C and finally stopped by adding 25 % formamide and heating it up to 65 for 10 min.
Detection of the amplificates ;
a) Gel electrophoresis 15 ~ll of the above-listed reaction mixture containing the amplified nucleic acids were added onto an 1.2 % agarose gel (according to Maniatis, T., - :` 21~2963 E.F. Fritsch and J. Sambrook, 1990. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York), then subject to electrophoresis (lO0 V, 3 h), and subsequently plotted onto a nylon membrane (Boehringer Mannheim) in a vacuum blot apparatus (manufactured by Phannacia).
b) Hybridization The membrane was prehybridized for 1 h at 45C in 5 x SSC, 1 % blocking reagent (Boehringer Mannheim), 0.1 % n-lauryl sarcosin (sodium salt) and 0.02 ~O SDS. For the hybridization procedure to occur, the membrane was incubated overnight at 45C with 100 ng digoxigenin-labeled Nas 4-probe per ml hybridization solution (see above).
Non-specifically bound probe was washed out as follows: ;
2 x with 2 x SSC; 0.2 % SDS at 20C
2 x with 0.1 x SSC; 0.1 % SDS at 45C
c) Detection After incubation with aLtcaline phosphatase-labeled anti-digoxigenin antibodies, the amplificates hybridized with digoxigenin-labeled NAS 4 probe were chemiluminometrically visualized with AMPPD (filrther details see DIG Luminescent Detection Kit, Boehringer Mannheim, Cat. No.
1363514).
Using a a Polaroid b/w film, the membrane was subject to a 5 min exposure. The result can be seen in Figure 7.
-" 2102963 SEQUENCE LISTlNG
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Boehringer Mannheim GmbH
(B) STREET: Sandhoferstr. 116 (C) CITY: Mannheim (E) COUNTRY: DE
(F) POSTAL CODE ~ZIP):68298 (G) TELEPHONE: 0621-759-4348 (H) TELEFAX: 0621-7S9-4457 (ii) TITLE OF INVENTION: Simple nucleic acid amplification process (iii) N~IBER OF SEQUENCES: 3 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patent~n Release #1.0, Version #1.25 (EPO) (2) INFORMATIQN FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS~
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~ ~ ::~. -(D)TOPOLOGY: linear ;~
(ii)MOLECULETYPE: cDNA
. ~.
- .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
' .
:~`` 2102~63 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
(2) lNFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULETYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Then the reaction vessel was clo~ed and not opened again until the amplification procedure was stopped. The reaction was allowed to occur for -~
90 min at 40C and finally stopped by adding 25 % formamide and heating it up to 65 for 10 min.
Detection of the amplificates ;
a) Gel electrophoresis 15 ~ll of the above-listed reaction mixture containing the amplified nucleic acids were added onto an 1.2 % agarose gel (according to Maniatis, T., - :` 21~2963 E.F. Fritsch and J. Sambrook, 1990. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York), then subject to electrophoresis (lO0 V, 3 h), and subsequently plotted onto a nylon membrane (Boehringer Mannheim) in a vacuum blot apparatus (manufactured by Phannacia).
b) Hybridization The membrane was prehybridized for 1 h at 45C in 5 x SSC, 1 % blocking reagent (Boehringer Mannheim), 0.1 % n-lauryl sarcosin (sodium salt) and 0.02 ~O SDS. For the hybridization procedure to occur, the membrane was incubated overnight at 45C with 100 ng digoxigenin-labeled Nas 4-probe per ml hybridization solution (see above).
Non-specifically bound probe was washed out as follows: ;
2 x with 2 x SSC; 0.2 % SDS at 20C
2 x with 0.1 x SSC; 0.1 % SDS at 45C
c) Detection After incubation with aLtcaline phosphatase-labeled anti-digoxigenin antibodies, the amplificates hybridized with digoxigenin-labeled NAS 4 probe were chemiluminometrically visualized with AMPPD (filrther details see DIG Luminescent Detection Kit, Boehringer Mannheim, Cat. No.
1363514).
Using a a Polaroid b/w film, the membrane was subject to a 5 min exposure. The result can be seen in Figure 7.
-" 2102963 SEQUENCE LISTlNG
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Boehringer Mannheim GmbH
(B) STREET: Sandhoferstr. 116 (C) CITY: Mannheim (E) COUNTRY: DE
(F) POSTAL CODE ~ZIP):68298 (G) TELEPHONE: 0621-759-4348 (H) TELEFAX: 0621-7S9-4457 (ii) TITLE OF INVENTION: Simple nucleic acid amplification process (iii) N~IBER OF SEQUENCES: 3 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patent~n Release #1.0, Version #1.25 (EPO) (2) INFORMATIQN FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS~
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~ ~ ::~. -(D)TOPOLOGY: linear ;~
(ii)MOLECULETYPE: cDNA
. ~.
- .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
' .
:~`` 2102~63 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
(2) lNFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULETYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Claims (15)
1. Process for amplifying a segment of a deoxy-ribonucleic acid A comprising the following steps:
a) Converting possibly present double-stranded deoxyribonucleic acid A into single strands resulting in formation of strands A- and A+, b) Formation of a strand B+ which is at least partially complementary to strand A- by extending a first primer P1+ containing a promoter-containing nucleotide sequence and a sequence which is specific for a partial sequence of the segment, c) Formation of a strand B- which is essentially complementary to strand B+ by extending a second primer P2-, the strands B- and B+
together containing a functional promoter for DNA-directed RNA polymerase in addition to sequence information from nucleic acid A, d) Transcription of a nucleic acid double strand of B- and B+ under control of said promoter to form a ribonucleic acid C+, e) Formation of a strand D'- which is essen-tially complementary to C+ by extending a primer, f) Formation of a strand D+ which is essentially complementary to D'- by extending a primer and extending D'- to D-, while the strands D-and D+ together contain a functional promoter in addition to sequence information of ribonucleic acid C+, g) Transcription of the nucleic acid double strand of D- and D+ under control of said promoter to form ribonucleic acid C+, characterized in that one or several reagents necessary for carrying out steps c to f are added to a reaction mixture prior to or simultaneously with formation of the nucleic acid B+.
a) Converting possibly present double-stranded deoxyribonucleic acid A into single strands resulting in formation of strands A- and A+, b) Formation of a strand B+ which is at least partially complementary to strand A- by extending a first primer P1+ containing a promoter-containing nucleotide sequence and a sequence which is specific for a partial sequence of the segment, c) Formation of a strand B- which is essentially complementary to strand B+ by extending a second primer P2-, the strands B- and B+
together containing a functional promoter for DNA-directed RNA polymerase in addition to sequence information from nucleic acid A, d) Transcription of a nucleic acid double strand of B- and B+ under control of said promoter to form a ribonucleic acid C+, e) Formation of a strand D'- which is essen-tially complementary to C+ by extending a primer, f) Formation of a strand D+ which is essentially complementary to D'- by extending a primer and extending D'- to D-, while the strands D-and D+ together contain a functional promoter in addition to sequence information of ribonucleic acid C+, g) Transcription of the nucleic acid double strand of D- and D+ under control of said promoter to form ribonucleic acid C+, characterized in that one or several reagents necessary for carrying out steps c to f are added to a reaction mixture prior to or simultaneously with formation of the nucleic acid B+.
2. Process for amplifying a segment of a deoxy-ribonucleic acid A comprising the following steps:
a) Converting a possibly present double-stranded deoxyribonucleic acid A into single strands resulting in formation of strands A- and A+, b) Formation of a strand B+ which is at least partially complementary to strand A- by extending a first primer P1+ containing a promoter-containing nucleotide sequence and a sequence which is specific for a partial sequence of the segment, c) Formation of a strand B- which is essentially complementary to strand B+ by extending a second primer P2-, the strands B- and B+
together containing a functional promoter for DNA-directed RNA polymerase in addition to sequence information from nucleic acid A, d.) Transcription of nucleic acid double strand of B- and B+ under control of said promoter to form a ribonucleic acid C+, e) Formation of a strand D'- which is essen-tially complementary to C+ by extending a primer, f) Formation of a strand D+ which is essentially complementary to D'- by extending a primer and extending D'- to D-, while the strands D-and D+ together contain a functional promoter in addition to sequence information of ribonucleic acid C+, g) Transcription of the nucleic acid double strand of D- and D+ under control of said promoter to form ribonucleic acid C+, characterized in that there is no complete separation between B-and A+ between steps b and c.
a) Converting a possibly present double-stranded deoxyribonucleic acid A into single strands resulting in formation of strands A- and A+, b) Formation of a strand B+ which is at least partially complementary to strand A- by extending a first primer P1+ containing a promoter-containing nucleotide sequence and a sequence which is specific for a partial sequence of the segment, c) Formation of a strand B- which is essentially complementary to strand B+ by extending a second primer P2-, the strands B- and B+
together containing a functional promoter for DNA-directed RNA polymerase in addition to sequence information from nucleic acid A, d.) Transcription of nucleic acid double strand of B- and B+ under control of said promoter to form a ribonucleic acid C+, e) Formation of a strand D'- which is essen-tially complementary to C+ by extending a primer, f) Formation of a strand D+ which is essentially complementary to D'- by extending a primer and extending D'- to D-, while the strands D-and D+ together contain a functional promoter in addition to sequence information of ribonucleic acid C+, g) Transcription of the nucleic acid double strand of D- and D+ under control of said promoter to form ribonucleic acid C+, characterized in that there is no complete separation between B-and A+ between steps b and c.
3. Process according to claim 1 or 2, characterized in that reagents necessary for carrying out said process include at least one reagent from the group consisting of RNA polymerase, primer P2-, and ribonucleoside triphosphates.
4. Process according to claim 1, characterized in that after addition of P1+ no further denaturation of nucleic acids occurs.
5. Process according to claim 1 or 2, characterized in that the primers have a nucleotide sequence selected such that primer P1+ hybridizes with nucleic acid A-, such that the 5'-end of a target-specific sequence is at least one nucleotide away from the 3'-end of said nucleic acid A- and located between said 3'-end and said 5'-end of said nucleic acid A-.
6. Process according to claim 1 or 2, characterized in that at its 5'-end, P1+ contains a sense strand of a promoter.
7. Process according to claim 1 or 2, characterized in that the nucleic acid A does not contain any promoter sequences.
8. Process according to claim 1 or 2, characterized in that the reagents contain an RNase H.
9. Process according to claim 1, characterized in that a denaturation between steps b and c does not occur.
10. Process for detecting deoxyribonucleic acids A
by means of - carrying out steps a-g according to claim 1, and - detecting the formed nucleic acids C+, D'-, D-or D+, or hybrids thereof.
by means of - carrying out steps a-g according to claim 1, and - detecting the formed nucleic acids C+, D'-, D-or D+, or hybrids thereof.
11. Reagent kit for amplifying nucleic acids containing - a description of a process according to claim 1, - a container 1 with an enzyme mixture containing an RNA polymerase, - a container 2 with two primers having different sense orientations, of which at least one contains a promoter sequence, and containing an enzyme with reverse transcriptase activity, - a container 3 containing deoxyribonucleotides and ribonucleotides, - a packaging.
12. Reagent kit for detecting nucleic acids containing - the containers 1-3 of the reagent kit according to claim 11, - a container 4 containing a nucleic acid probe, - a description of the process according to claim 10, and - the packaging.
13. The reagent kit according to claim 12, wherein additives are present in one or several suitable containers, while the contents of one or several of said containers are combined.
14. Set of oligonucleotides for detecting bacteria of the family Listeria containing at least two Listeria-specific primers, a first and second primer, which essentially do not hybridize with themselves under conditions of hybridization of oligonucleotides with nucleic acids from Listeria, said primers being such that an extension product of said first primer can serve as a template for the extension of said second primer.
15. Set according to claim 14, characterized in that in addition it contains a Listeria-specific probe, which can hybridize in a segment of an extension product of said two primers which is located between said primers.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4238699A DE4238699A1 (en) | 1992-11-17 | 1992-11-17 | Simple nucleic acid amplification method |
| DE4238699.3 | 1992-11-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2102963A1 true CA2102963A1 (en) | 1994-05-18 |
Family
ID=6473038
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002102963A Abandoned CA2102963A1 (en) | 1992-11-17 | 1993-11-12 | Simple nucleic acid amplification |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0598332A2 (en) |
| JP (1) | JPH06209776A (en) |
| AU (1) | AU5057093A (en) |
| CA (1) | CA2102963A1 (en) |
| DE (1) | DE4238699A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5922538A (en) * | 1996-11-08 | 1999-07-13 | E.I. Du Pont De Nemours And Company | Genetic markers and methods for the detection of Listeria monocytogenes and Listeria spp |
| US6312930B1 (en) | 1996-09-16 | 2001-11-06 | E. I. Du Pont De Nemours And Company | Method for detecting bacteria using PCR |
| US6312928B1 (en) | 1997-11-17 | 2001-11-06 | Akzo Nobel N.V. | Transcription based amplification of double stranded DNA targets |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995015399A1 (en) | 1993-12-01 | 1995-06-08 | Toyo Boseki Kabushiki Kaisha | Method of amplifying and detecting target nucleic acid sequence by using thermostable enzymes |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU624601B2 (en) * | 1988-01-21 | 1992-06-18 | Genentech Inc. | Amplification and detection of nucleic acid sequences |
| DE69034177T2 (en) * | 1989-07-11 | 2005-10-27 | Gen-Probe Inc., San Diego | Process for the amplification of nucleic acid sequences |
-
1992
- 1992-11-17 DE DE4238699A patent/DE4238699A1/en not_active Withdrawn
-
1993
- 1993-11-10 AU AU50570/93A patent/AU5057093A/en not_active Abandoned
- 1993-11-11 EP EP93118263A patent/EP0598332A2/en not_active Withdrawn
- 1993-11-12 CA CA002102963A patent/CA2102963A1/en not_active Abandoned
- 1993-11-16 JP JP5286585A patent/JPH06209776A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6312930B1 (en) | 1996-09-16 | 2001-11-06 | E. I. Du Pont De Nemours And Company | Method for detecting bacteria using PCR |
| US5922538A (en) * | 1996-11-08 | 1999-07-13 | E.I. Du Pont De Nemours And Company | Genetic markers and methods for the detection of Listeria monocytogenes and Listeria spp |
| US6312928B1 (en) | 1997-11-17 | 2001-11-06 | Akzo Nobel N.V. | Transcription based amplification of double stranded DNA targets |
Also Published As
| Publication number | Publication date |
|---|---|
| DE4238699A1 (en) | 1994-05-19 |
| AU5057093A (en) | 1994-06-16 |
| EP0598332A2 (en) | 1994-05-25 |
| JPH06209776A (en) | 1994-08-02 |
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