EP1327004A2 - Procede de detection d'une sequence d'acide nucleique - Google Patents

Procede de detection d'une sequence d'acide nucleique

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Publication number
EP1327004A2
EP1327004A2 EP01982109A EP01982109A EP1327004A2 EP 1327004 A2 EP1327004 A2 EP 1327004A2 EP 01982109 A EP01982109 A EP 01982109A EP 01982109 A EP01982109 A EP 01982109A EP 1327004 A2 EP1327004 A2 EP 1327004A2
Authority
EP
European Patent Office
Prior art keywords
identification
sequence
reaction
sequences
primers
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.)
Withdrawn
Application number
EP01982109A
Other languages
German (de)
English (en)
Inventor
Jürgen SCHÜLEIN
Hans Kosak
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
November AG Novus Medicatus Bertling Gesellschaft fuer Molekular Medizin
Original Assignee
November AG Novus Medicatus Bertling Gesellschaft fuer Molekular Medizin
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by November AG Novus Medicatus Bertling Gesellschaft fuer Molekular Medizin filed Critical November AG Novus Medicatus Bertling Gesellschaft fuer Molekular Medizin
Publication of EP1327004A2 publication Critical patent/EP1327004A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the invention relates to a method for the detection of at least one nucleic acid sequence and a kit for carrying out the method according to the invention.
  • PCR polymerase chain reaction
  • the duplicated sequences can then be detected by means of hybridization with a binding sequence specific for the nucleic acid sequence to be detected. It is disadvantageous in this method that a specific binding sequence is required for each nucleic acid sequence to be detected. Furthermore, it is disadvantageous that specific hybridization conditions must be maintained for each hybridization with this binding sequence.
  • the primer has a region in which the synthesis of a counter strand by a blocking agent for a polymerase is prevented.
  • the area has a fluorophore and a quencher, which are located in the immediate vicinity due to base pairings. A fluorescence signal cannot be generated.
  • the region has a sequence which is complementary to a product resulting from the extension of the primer during the PCR. After denaturation of the product, base pairing of the section with the complementary region in the product occurs under suitable conditions.
  • the quencher causes the quencher to be separates so that a fluorescence signal can arise.
  • the fluorescence signal serves as evidence of the presence of a DNA sequence specific for the primer.
  • the method has the disadvantage that a primer specific for each nucleic acid sequence to be detected has to be synthesized with a specific complementary section. Due to the various functional units, the primer is relatively long and therefore difficult to manufacture.
  • nucleotides in a PCR which do not occur in the nucleic acid sequence to be reproduced.
  • a nucleotide can be deoxy uridine.
  • an undesirable product can be treated with uracil DNA glycosylase.
  • Uracil DNA glycolyase cleaves the gycosidic bond between the base uracil and the sugar deoxy-ribose of a deoxy-uridine residue built into a DNA molecule.
  • the undesired product can no longer serve as a template in a further replication reaction.
  • Another nucleotide that can be used is bromodeoxy uridine. DNA containing bromo-deoxy-uridine can be degraded by treatment with light under suitable conditions.
  • No. 5,744,311 discloses a amplification process for nucleic acids based on strand displacement.
  • a primer is hybridized with the 3 'end of a single-stranded nucleic acid to be amplified and extended by means of a DNA polymerase.
  • Deoxy nucleoside triphosphates are used for the extension, some of which are derivatized.
  • Suitable derivatized deoxy nucleoside triphosphates are, for example, ⁇ -thio-deoxy nucleoside triphosphates.
  • a strand of the resulting double-stranded DNA with derivatized deoxy nucleotide residues is len recognized by a restriction endonuclease and cut. Starting from the cut, the DNA polymerase extends the 3 'end of the cut DNA strand. The other part of the cut DNA strand is displaced by the double-stranded DNA. The displaced DNA strand is multiplied by repeating the process.
  • WO 97/31256 it is known to detect target sequences in nucleic acids by means of a ligase reaction and an addressable matrix having immobilized binding sequences.
  • an oligonucleotide probe is used which has a target sequence-specific and a binding sequence-specific portion.
  • a second oligonucleotide probe which has a labeling substance, is used, which can bind to the target sequence to be detected in the immediate vicinity of the first oligonucleotide probe. If the target sequence is present, the first and second oligonucleotide probes bind to the target sequence. They are covalently connected to each other by a ligase.
  • Probes are brought into contact with the immobilized binding sequences so that hybridization takes place.
  • the presence of the target sequence is detected by detecting the labeling substance at the hybridization site.
  • the disadvantage of the method is that it often gives false positive results.
  • US 5,525,494 describes a method for amplifying a target nucleotide sequence, a first primer being used.
  • the first primer has a binding section that is essentially complementary to the target nucleotide sequence.
  • An extension section is tied to this.
  • the extension section is designed so that a synthesis of a complementary extension section is suppressed.
  • the extension section formed at the 5 'end remains single-stranded.
  • the products formed in the polymerase chain reaction (PCR) can thus be bound to suitable oligonucleotides immobilized on a solid phase.
  • the detection sensitivity of the known method is not particularly high.
  • the object of the present invention is to eliminate the disadvantages of the prior art.
  • a universal method and a kit for the parallel detection of nucleic acid sequences are to be specified.
  • a method for the parallel detection of nucleic acids using a section-wise single-stranded nucleic acid reaction is provided with the following steps:
  • a) Providing compounds each formed from a first primer specific for the nucleic acids to be detected and an identification sequence specific to the first primers, the identification sequence being selected from a group of identification sequences which, given predetermined uniform hybridization conditions, do not crosshy among one another and not with the first primers - bridize, and wherein in the connection a means is provided that the single-strandedness of a section of the lit. c product formed, b) bringing the nucleic acids into contact with the compounds and with second primers specific for the nucleic acids to be detected, the second primers being selected from a group of second primers which do not cross-hybridize with the identification sequences under predetermined uniform hybridization conditions,
  • binding sequences are selected from a group of binding sequences which do not cross-hybridize with incompletely complementary single-stranded sections of the specific products under given uniform hybridization conditions, and
  • a primer is understood to be an oligonucleotide that can be extended by a polymerase.
  • the primers can consist of DNA.
  • First and second primers specific for the nucleic acid sequence to be detected are primers which hybridize specifically under suitable hybridization conditions with the nucleic acid sequence to be detected or with a complementary complementary strand.
  • An identification sequence specific for the first primer is an identification sequence which is clearly assigned to the first DNA primer. The agent can be contained within the identification sequence, within the first primer or between the first primer and the identification sequence.
  • a specific product is understood to mean a product which is specific for the nucleic acid sequence to be detected. Such a product is in step lit. c formed especially under stringent conditions.
  • step lit. e the specific hybridization of the identification sequence or of the single-stranded section with the binding sequence takes place under suitable stringent conditions. These conditions depend on the identification sequences or the binding sequences.
  • the advantage of the method is that the specific product formed can hybridize directly with the immobilized binding sequence without being previously denatured. This prevents the single strands forming the double-stranded section of the specific product from interfering with the hybridization with the binding sequences, for example through cross-hybridization. It is also advantageous in the method that single-stranded regions hybridize with the binding sequences when the products are hybridized. Egg- Hybridization between single-stranded sections is considerably faster and more efficient compared to hybridization between denatured double-stranded sections. It is furthermore advantageous that the carrier with a set of immobilized binding sequences can be used for the detection of different nucleic acid sequences. Predefined identical identification sequences can be linked to different first primers in different detection methods.
  • Identical binding sequences can then be used for the detection of different nucleic acid sequences.
  • the method according to the invention can be used universally for the detection of nucleic acids. Because of the proposed selection of primer identification and binding sequences, any number of nucleic acids can be detected in a parallel method. This property is also called multiplexing.
  • the proposed selection of the first and second primers has the effect that the formation of primer dimers is reduced.
  • Primer dimers would be used in step lit. e lead to false positive results and the amount of specific products formed in step lit.
  • Reduce c Since it is excluded according to a further provision of the invention that the first primers hybridize with the identification sequences, it is prevented that the amount of the first primers required for the formation of the specific product is reduced. It also prevents the amount of single-stranded sections required to form the specific product from being reduced.
  • the multiplexing ability requires specific properties of the second primer used, the identification sequences and the binding sequences.
  • the second primer should consist of A group of second primers can be selected, which specifically bind to their complementary sequences on the nucleic acids to be detected under a hybridization condition that applies to all primers. Furthermore, the second primers are selected so that under the hybridization conditions according to lit. c no cross-hybridization with one another and no hybridization of the primers with the identification sequences takes place. It is also advisable to select the second primer so that under the conditions of step lit. e Cross hybridization of the second primer with the binding sequences is excluded. This is particularly useful if in step lit. d free primers are not separated.
  • the selection of the identification sequences has the advantage that the amount of the lit. c products formed increases and at the same time the amount of non-specific products is reduced.
  • the single-stranded sections of the products can be selected from a group of single-stranded sections, which at least under the conditions of step lit. e do not hybridize with each other. Hybridization would be disadvantageous in this case because the concentration of those for the binding of the single-stranded sections to the binding sequences would be reduced.
  • the binding sequences are expediently selected from a group of binding sequences which, at least under the conditions of step lit. hybridize specifically with the complementary, single-stranded sections of the products. They do not hybridize to non-complementary single-stranded sections of the products. A hybridization of the binary Ending sequences with incompletely complementary binding sequences of the products would lead to false positive results or at least a lower detection sensitivity of the nucleic acids to be detected.
  • the first primers can also be selected from a group of first primers which do not cross-hybridize under given uniform hybridization conditions.
  • the compounds can contain an agent which, during the reaction, alters a synthesis of a counter strand complementary to the identification sequence.
  • the compounds can contain an agent which, during the reaction, enables the identification sequence or its complementary counter-strand to be degraded.
  • the agent can e.g. a uracil, a thionucleotide and / or a ribonucleotide.
  • the degradation reaction can be carried out using uracil DNA glycosylase, exonuclease, RNAseH, under the action of light or by means of a restriction endonuclease which cleaves the identification sequence or its counter strand, in particular BsrI, BstNI, BsmAI, BsII, BsoBI or BstOI.
  • a degradation reaction is understood to mean a reaction which modifies the identification sequence or its counter strand. It separates the identification sequence or the counter strand from the respectively unmodified complex mental strand.
  • the degradation reaction can be a single bond in the identification sequence or the counter-cleavage reaction. Such a reaction can lead to fragmentation of the identification sequence or the opposite strand.
  • the hybrid of the identification sequence and the counter strand is thus destabilized thermodynamically so that it is the latest among those for step lit. e required stringent conditions dissociated.
  • the identification sequence can be complementary in the terminal areas. It can have a refolding, preferably having 4 to 10 base pairs. This increases the stringency of the hybridization in step lit. e.
  • the reaction is carried out with the formation of conditions which counteract primer dimers. This helps prevent false positive results.
  • the conditions are chosen so that the first and second primers do not hybridize with one another in the reaction producing the product.
  • the primers can be formed from a group of primers in which the melting point of the cross hybrids is at least 10 ° C. below the lowest melting point of a specific hybrid of a primer with the nucleic acid to be detected. Furthermore, the primers can be selected from a group of primers in which cross hybrids of primers and identification sequences are at least 10 ° C. below the lowest melting point of a specific hybrid of a primer. The aforementioned are advantageously located
  • the reaction can be a primer extension reaction, preferably a polymerase chain reaction (PCR) or a strand displacement reaction.
  • PCR polymerase chain reaction
  • hot-start conditions are selected as conditions in the primer extension reaction. This counteracts the formation of primer dimers and other non-specific products.
  • the nucleic acid sequence to be detected can be a DNA or an RNA. If it is an RNA, the connection can additionally be brought into contact with a reverse transcriptase and in step lit. c in addition, reverse transcription is carried out. In step lit. b in addition, a third primer, which is specific for the nucleic acid sequence to be detected, on which step lit. c nucleotides are added using the reverse transcriptase.
  • an optically or electrically detectable marking can be built into the product during the reaction.
  • the second primer can have a preferably 5 'terminal labeling group, or it can be lit.
  • c labeled nucleotides are inserted into the product by polymerization. Suitable markings are e.g. fluorescent nucleotides, biotin, hapten or redox markers.
  • the melting point of the double-stranded section of the product is expediently greater than the hybridization temperature. rature of a hybrid formed from the single-stranded section of the product with the binding sequence.
  • the binding sequence can be complementary in the terminal areas. It can have a refolding, preferably having 4 to 10 base pairs. This increases the stringency of the hybridization of the binding sequences with the single-stranded regions of the products.
  • the products are separated from non-extended connections.
  • the separation can be done using glass / silica particles or a filter.
  • identification sequences in particular are removed. This significantly increases the sensitivity of the method to detection.
  • the identification sequences can be destroyed in a targeted manner after the formation of the specific products. This is possible, for example, when using uracil in the identification sequences and when treating with uracil glycolysis when using ribonucleotides in the identification sequences.
  • Such treatment is expediently carried out in an alkaline environment. In this case the environment is then neutralized again in order to ensure renaturation to the double strand of the product before contact with the binding sequences.
  • a kit for carrying out the method according to the invention, comprising
  • the kit can also contain activated identification sequences which are suitable for coupling to 5 'modified primers.
  • the identification sequence can be selected from a group of identification sequences which do not cross-hybridize under given uniform hybridization conditions.
  • the end of an identification sequence can have a maleiimide or succiimide group. These groups enable simple coupling of the identification sequence to a primer modified with a thiol or amine group.
  • the advantage of such a kit is that a user of the method can use the same set of immobilized binding sequences and identification sequences for each of his specific nucleic acids to be detected. Because of the further advantageous refinements of the kit, reference is made to the preceding explanations.
  • the features mentioned there can also be features and / or components of the kit.
  • FIG. 1 a, b a schematic representation of the duplication of a nucleic acid sequence to be detected by means of a PCR
  • FIG. 2 a - c a schematic representation of binding sequences immobilized on a support and their hybridization with (FIG. 2 b) and without
  • FIG. 3 shows a schematic representation of the multiplication of an RNA by means of a reverse transcriptase and a DNA polymerase in one
  • FIG. 4 shows a schematic representation of a duplication of a nucleic acid sequence to be detected by means of a PCR and a subsequent degradation reaction.
  • FIG. 1 a shows a compound 14 composed of a first primer 10 and an identification sequence 12.
  • the primer 10 binds to the nucleic acid sequence 16 to be detected.
  • the second primer 18 has a labeling substance 20. It binds to the opposite strand of the nucleic acid sequence 16 to be detected. the result of a PCR is shown, which has been carried out with the compound 14 shown in FIG. 1 a, the second primer 18 and the nucleic acid sequence 16 to be detected.
  • the identification sequence 12 contains means which prevent the region of the identification sequence 12 from becoming double-stranded. Products with a double-stranded section and the single-stranded identification sequence were created.
  • Fig. 2a shows a carrier 22. This can e.g. act as a membrane. Binding sequences 26 are bound to the carrier 22 via linkers 24. 2b shows the situation after the binding sequences 26 shown in FIG. 2a have been brought into contact with the single-stranded sections of the products. The hybridization can be detected on the carrier 22 by the marking substance. 2c shows, by comparison, a situation in which non-elongated connections have not been removed prior to contacting the product with the binding sequences 26. The simultaneous presence of product and free compounds can lead to competition for the specific binding sequences on the carrier 22. Because of the greater mobility, size, charge density, and number of free compounds compared to the product, binding of the free compound is the preferred reaction with the binding sequences 26.
  • FIG. 3 schematically shows a method in which the nucleic acid sequence 16 to be detected is an RNA. First, the second one containing the marking substance 20 binds
  • Primer 18 to the RNA It is extended using a reverse transcriptase. This creates a double-stranded DNA-RNA hybrid. After denaturing the DNA-RNA hybrid the first primer 10 binds to the DNA strand. A PCR is carried out to reproduce the DNA strand.
  • the 4 shows a compound 14 consisting of a primer 10 and an identification sequence 12.
  • the identification sequence 12 has nucleotides 13 which can be cleaved by a degradation reaction.
  • a PCR reaction with this compound, a nucleic acid sequence 16 to be detected and a second primer 18 leads to completely double-stranded PCR products.
  • the identification sequences contained therein are removed by a degradation reaction. The result is double-stranded products with a single-stranded counter strand of the identification sequence.

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  • Chemical & Material Sciences (AREA)
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  • Life Sciences & Earth Sciences (AREA)
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  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Kit permettant de mettre en oeuvre un procédé, qui contient (a) des séquences de liaison immobilisées sur un substrat et (b) des liaisons formées chacune d'une première amorce spécifique des acides nucléiques à détecter et d'une séquence d'identification spécifique de la première amorce. La séquence d'identification est choisie dans un groupe de séquences d'identification qui ne s'hybrident pas de manière croisée dans des conditions d'hybridation homogènes prédéterminées.
EP01982109A 2000-09-18 2001-09-18 Procede de detection d'une sequence d'acide nucleique Withdrawn EP1327004A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10046184 2000-09-18
DE10046184A DE10046184A1 (de) 2000-09-18 2000-09-18 Verfahren zum Nachweis mindestens einer Nukleinsäuresequenz
PCT/DE2001/003564 WO2002024944A2 (fr) 2000-09-18 2001-09-18 Procede de detection d'une sequence d'acide nucleique

Publications (1)

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EP1327004A2 true EP1327004A2 (fr) 2003-07-16

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EP01982109A Withdrawn EP1327004A2 (fr) 2000-09-18 2001-09-18 Procede de detection d'une sequence d'acide nucleique

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EP (1) EP1327004A2 (fr)
DE (1) DE10046184A1 (fr)
WO (1) WO2002024944A2 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10253337B4 (de) * 2002-11-14 2005-10-20 November Ag Molekulare Medizin Verfahren zum Nachweis einer Nukleinsäure
US20050026181A1 (en) * 2003-04-29 2005-02-03 Genvault Corporation Bio bar-code
FR2949120A1 (fr) * 2009-08-13 2011-02-18 Centre Nat Rech Scient Procede de detection d'un adn circularise et utilisation de ce procede pour la detection de mutations
US9175339B2 (en) 2009-10-29 2015-11-03 Ngk Insulators, Ltd. Method for detection of target nucleic acid
JPWO2012070618A1 (ja) * 2010-11-24 2014-05-19 株式会社カネカ 増幅核酸検出方法及び検出デバイス
SG11201400635UA (en) * 2011-09-14 2014-09-26 Ngk Insulators Ltd Method for detecting target nucleic acid
US9783844B2 (en) 2012-04-27 2017-10-10 Kaneka Corporation Method for amplifying nucleic acid and method for detecting amplified nucleic acid
US10392652B2 (en) 2013-11-22 2019-08-27 Kaneka Corporation Micro RNA detection method using two primers to produce an amplified double stranded DNA fragment having a single stranded region at one end
AU2018400335A1 (en) * 2018-01-05 2020-07-02 Quotient Suisse Sa Self-assembling diagnostic array platform

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Publication number Priority date Publication date Assignee Title
GB8920097D0 (en) * 1989-09-06 1989-10-18 Ici Plc Amplification processes
EP0541722B1 (fr) * 1990-08-03 1995-12-20 Sterling Winthrop Inc. Composes et procedes d'inhibition de l'expression de genes
US5518900A (en) * 1993-01-15 1996-05-21 Molecular Tool, Inc. Method for generating single-stranded DNA molecules
US5648211A (en) * 1994-04-18 1997-07-15 Becton, Dickinson And Company Strand displacement amplification using thermophilic enzymes
US5830655A (en) * 1995-05-22 1998-11-03 Sri International Oligonucleotide sizing using cleavable primers
US6090553A (en) * 1997-10-29 2000-07-18 Beckman Coulter, Inc. Use of uracil-DNA glycosylase in genetic analysis

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Title
See references of WO0224944A2 *

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Publication number Publication date
WO2002024944A3 (fr) 2003-05-08
WO2002024944A2 (fr) 2002-03-28
DE10046184A1 (de) 2002-04-04

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