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WO1996005296A1 - Procede de preparation et d'amplification d'acides nucleiques - Google Patents

Procede de preparation et d'amplification d'acides nucleiques Download PDF

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Publication number
WO1996005296A1
WO1996005296A1 PCT/DE1995/001003 DE9501003W WO9605296A1 WO 1996005296 A1 WO1996005296 A1 WO 1996005296A1 DE 9501003 W DE9501003 W DE 9501003W WO 9605296 A1 WO9605296 A1 WO 9605296A1
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Prior art keywords
molecule
starter
nucleotide sequence
template
template molecule
Prior art date
Application number
PCT/DE1995/001003
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German (de)
English (en)
Inventor
Karl-Otto Greulich
Dino Celeda
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Institut Für Molekulare Biotechnologie, E.V.
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.)
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Publication date
Application filed by Institut Für Molekulare Biotechnologie, E.V. filed Critical Institut Für Molekulare Biotechnologie, E.V.
Priority to AU31604/95A priority Critical patent/AU3160495A/en
Publication of WO1996005296A1 publication Critical patent/WO1996005296A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • This invention relates to a method for producing and / or amplifying nucleic acids without the addition of defined starters (“primers”), the use of these nucleic acids and a kit containing the nucleic acids produced according to the invention.
  • Double-stranded nucleic acid sequences can be amplified by adding starters specific to each of the strands with an extension reaction induced by the starters using nucleotides and an enzyme suitable for the extension reaction.
  • This reaction referred to as the polymerase chain reaction ("PCR")
  • PCR polymerase chain reaction
  • primers functioning as starters is required in order to obtain the desired amplification of the double-stranded nucleic acid sequences.
  • each strand induced and enzymatically synthesized by one of the specific starters forms the template for a strand induced by the other starter and to be synthesized enzymatically, the enzymatically synthesized strands of a cycle each being complementary nucleic acid sequences.
  • the reaction cycles can be repeated any number of times until a desired amount of the double-stranded nucleic acid is present in the reaction mixture.
  • a disadvantage of PCR is the additional addition of sequence-specific oligonucleotide primers, which presupposes that there must be complementary sequences corresponding to these oligonucleotide sequences in the nucleic acid molecules to be amplified.
  • amplification of the primer by self-attachment can occur in the PCR, which results in an incorrect reaction product and incorrect signals in the case of a subsequent in situ hybridization with the amplified nucleic acid sequences.
  • the likelihood of contamination with foreign nucleic acids is increased in the reaction mixture for the PCR, since two additional pipetting steps are necessary due to the additional addition of the starters.
  • This object is achieved according to the invention by a method for producing and / or amplifying nucleic acids, the reaction mixture comprising a single-stranded nucleic acid molecule ("starter molecule”) with a terminal, preferably 3 '-terminal nucleotide sequence acting as a starter and a single-stranded nucleic acid moiety lekül ("template molecule”) with at least one nucleotide sequence capable of attachment to the terminal nucleotide sequence of a starter molecule, comprising the steps
  • nucleic acid molecule means a native, semi-synthetic, synthetic or modified nucleic acid molecule composed of deoxyribonucleotides and / or ribonucleotides and / or modified nucleotides, such as amino nucleotides or [cu-S] triphosphate nucleotides.
  • starter molecule means a nucleic acid molecule defined above with at least one terminal, preferably 3 'terminal nucleotide sequence and a nucleotide sequence flanking the terminal nucleotide sequence, which preferably contains at least one further nucleotide sequence capable of attachment to a nucleotide sequence of the template molecule; see. Figures 1 to 7.
  • this further nucleotide sequence is located at the other end of the starter molecule, preferably at the 5 'end.
  • template molecule means a nucleic acid molecule defined above with at least one nucleotide sequence capable of being attached to the terminal nucleotide sequence of the starter molecule; see. Figures 1 to 7.
  • the template molecule contains at least one terminal, preferably 3 'terminal nucleotide sequence which is capable of attachment to the terminal nucleotide sequence of the starter molecule.
  • reaction mixture means a reaction mixture which, in addition to the nucleotides and at least one agent suitable for the synthesis of the extension product, contains one or more starter molecules and one or more template molecules, it being possible for there to be further nucleic acids which are not involved in the process according to the invention.
  • the starter molecules and / or the template molecules are in a sufficient concentration, preferably at least about lxlO -15 g, in Re ⁇ action approach before.
  • attachment means the formation of, for example, hydrogen bonds between single-stranded, complementary regions of nucleic acid molecules, in particular between the nucleotide sequences of the starter molecules and template molecules defined according to the invention, at a suitable temperature, preferably 90 ° C. or less, and if appropriate ⁇ at a suitable salt concentration, preferably 50 to 300 mM.
  • extension product means a “synthesized” nucleic acid sequence that is covalently bound to the terminal nucleotide sequence of the starter molecule via, for example, a phosphodiester, thioester or amide bond, the primary sequence of which is complementary to the corresponding sequence of the template molecule.
  • an agent suitable for the synthesis of the extension product means a native enzyme or a synthetic agent which acts as a catalyst in the synthesis of the extension product.
  • native enzymes are Taq polymerase, the Klenow fragment of DNA polymers I, E. coli DNA polymerase I and reverse transcriptase.
  • reaction product means a nucleic acid containing the extension product, the reaction product per se with each repetition of the reaction sequence (a) to (c) can be used as a starter molecule and / or as a template molecule according to the definitions given above.
  • the starter molecule and the template molecule are the same, the matrix molecule (or the starter molecule) containing at least one nucleotide sequence which, for attachment to the terminal, preferably 3 'terminal nucleotide sequence of the starter molecule ( or the template molecule) is capable; see. Figure 3.
  • the template molecule (or the starter molecule) can contain a further nucleotide sequence which is capable of attaching the terminal, preferably 3 'terminal nucleotide sequence of the extension product.
  • the starter molecule and the template molecule are the same and the template molecule (or the starter molecule) contains at least one nucleotide sequence located at the other end, preferably 5 'end, which is used for attachment to the terminal, preferably 3' end nucleotide sequence Starter molecule (or the matrix molecule) is capable; see. Figure 4.
  • the template molecule at least partially contains the complementary sequence of the starter molecule, the template molecule containing at least one, preferably two, nucleotide sequences capable of attachment to the terminal nucleotide sequence of the starter molecule; see. FIG. 5.
  • at least one of the nucleotide sequences of the template molecule capable of attachment to the terminal nucleotide sequence of the starter molecule is located at the end, preferably 5 'end.
  • the template molecule is the complementary sequence of the starter molecule T, at least two, preferably the same, being attached to the terminal nucleotide sequence of the starter molecule. nucleotide sequences capable of term molecule are finally localized; see. Figure 6.
  • the starter molecule is covalently bound to the template molecule, for example via a phosphodiester, thioester or amide bond, so that at least one nucleic acid is present in the reaction mixture of the process according to the invention, which comprises the primary sequences of the starter molecule and of the matrix molecule according to the definitions given above.
  • the terminal, preferably 3 'terminal nucleotide sequence of this nucleic acid is the nucleotide sequence of the starter molecule capable of attachment to at least one nucleotide sequence contained in the template molecule; see. FIG. 7.
  • the nucleotide sequence contained in the matrix molecule can be 3'- or 5'-terminal, for example.
  • the nucleotide sequences of the starter molecule and / or of the template molecule which are capable of attachment are preferably repetitive sequences.
  • the term “repetitive sequences” means repeating sequences, a distinction being made between (1) repetitive genes, such as genes from rRNA, tRNA, histones and immunoglobulins, (2) medium repetitive sequences consisting of about 200 to 300 Nucleotides, and (3) highly repetitive sequences, consisting of short sequences of at least about 20 bp, which can be repeated 100 times and, like the "Alu family" of eukaryotes (sequences of 300 bp), are distributed over the entire genome.
  • nucleotide sequences of the starter molecule and / or the template molecule capable of attachment can contain at least one recognition sequence for endonucleases or for other nucleic acid-cleaving agents, such as "molecular scissors", which are based on the formation of triple helix DNA recognition sequences.
  • part of the nucleotides can be marked in the reaction mixture his.
  • Suitable labels are, for example, nucleotides coupled with biotin, digoxigenin or fluorescent dyes or nucleotides labeled with a radioactive isotope.
  • the reaction products themselves can be labeled, for example by incorporating labeled nucleotides using "nick translation".
  • the protruding strand of the template molecule is connected to a genetic, carcinogenic or infectious disease.
  • a genetic, carcinogenic or infectious disease there are specific nucleic acid sequences which, modified or native or directly or indirectly, cause the induction of such a disease.
  • the method according to the invention can be carried out with single-stranded or double-stranded nucleic acids, double-stranded nucleic acids being converted into single-stranded nucleic acids according to methods known in the art, such as heat denaturation or pH-dependent denaturation with HC1 or NaOH, before step (a) become.
  • the reaction mixture has a suitable volume, for example 20 to 200 ⁇ l, and contains (l) a concentration of desired nucleotides suitable for the synthesis of the extension product, preferably 5 to 100 nmol, more preferably about 20 nmol, (2) for the synthesis units of a synthetic agent sufficient for the extension product, for example 1 to 15 units, preferably 5 units of Taq polymerase, and (3) at least about 10 ⁇ 15 g of starter molecules and template molecules or nucleic acids which comprise the starter molecule and the template molecule contain covalently linked together in a suitable reaction solution.
  • the reaction solution preferably contains MgCl 2 (1 to 200 mmol, preferably 1 to 50 mmol, more preferably 1 to 20 mmol and most preferably 3 mmol), NaCl (30 to 300 mmol, preferably 50 to 250 mmol, more preferably 100) up to 200 mmol and most preferably 160 mmol) and / or KC1 (10 to 70 mmol, preferably 30 to 70 mmol, more preferably 40 to 60 mmol and most preferably 50 mmol) and / or tris (hydroxymethyl) aminomethane (5 to 50 mmol, preferably 5 to 30 mmol, more preferably 5 to 20 mmol and most) preferably 10 mmol) and / or Tween 20 (polyoxyethylene sorbitan monolaurate) (0.01 to 0.1% by volume, preferably 0.01 to 0.06% by volume, more preferably 0.01 to 0.04% by volume) % and most preferably 0.02% by volume) and optionally gelatin (0.1 to 1 mmol).
  • step (a) of the method according to the invention the terminal nucleotide sequences of the starter molecules attach themselves to nucleotide sequences contained in the template molecules, with the formation of protruding strands which act as a template.
  • this addition is also referred to as "offset renaturation". The addition takes place at a temperature which depends in each case on the type of nucleic acids present in the reaction mixture.
  • the attachment of the nucleic acid molecules which originate from chromosomal DNA with medium repetitive and highly repetitive sequences is carried out at a temperature between 70 and 90 ° C.
  • the addition can take place in such a way that, according to FIG. 7, the single-stranded nucleic acid with, for example, “aluminum sequences” containing “inverted repeats”, forming a loop with the terminal, preferably 3′-terminal aluminum sequence to one in the nucleic acid contained complementary "Alu sequence” hybridized, the terminal "Alu sequence being in the region of the starter molecule and the complementary" Alu sequence being in the region of the template molecule according to the definitions given above.
  • a terminal "Alu sequence" in step (b) of the process according to the invention can be used to synthesize an extension Induct product by, for example, the Taq polymerase, where the protruding strand of the single-stranded nucleic acid is used as a matrix.
  • the temperature of the attachment or "renaturation temperature” is important. Repetitive sequences renaturate faster due to their frequent occurrence and their sometimes large AT content. Highly repetitive DNA already renatures at temperatures below 90 ° C. To a large extent, the renaturation is unspecific, whereby an offset ("hybridization") of the individual strands is partially achieved. This makes use of the present invention.
  • the rapid renaturation of the single-stranded strands or the rapid renaturation of a single-stranded nucleic acid with the formation of loops means that the so-called "annealing step", which is required for the PCR, is unnecessary in the production of nucleic acids from chromosomal DNA according to the invention .
  • the result of this is that the "single copy" genes contained in the chromosomal nucleic acids renaturate significantly more slowly and therefore do not renaturate, or only renaturate much more slowly under the selected conditions of, for example, above 70 ° C. and therefore cannot form start sequences for elongation.
  • the addition is carried out at a temperature of approximately 40 to 80 ° C. This enables individual, at times terminal AT-rich or GC-rich regions of the starter molecules, on complementary nucleotide sequences either within the single-stranded nucleic acids renaturation with loop formation or offset, the addition step to avoid a complete renaturation of the nucleic acids to be amplified should not exceed a duration of one hour.
  • step (a) of the method according to the invention can be applied mutatis mutandis to the embodiment in which the starter molecule and the template molecule are the same.
  • step (b) the synthesis of the elongation product ("elongation") is carried out at a temperature which depends in particular on the agent suitable for the synthesis, reaction products being obtained as defined above.
  • the synthesis takes place when the Taq polymerase is used at a temperature between 70 to 80 ° C., preferably 72 ° C., for 1 to 15 minutes, preferably 5 minutes.
  • step (c) the reaction product is separated from the matrix molecule, for example by heating the reaction mixture (“denaturing”) to 90 to 100 ° C., preferably 95 ° C., for i to 15 minutes, preferably 5 minutes.
  • the reaction sequence (a) to (c) is repeated at least once in step (d) of the process according to the invention.
  • the process according to the invention is repeated 1 to 200 times, preferably 80 times, with additional units of the agent suitable for the synthesis, for example 1 to 15 units, preferably 5 units of Taq polymerase, being added after every 40th repetition become.
  • reaction products produced by the process according to the invention can be cleaved at least once physically (e.g. by ultrasound) and / or chemically (e.g. by "molecular scissors") and / or enzymatically (e.g. by endonucleases) using suitable processes and / or means.
  • Another object of the present invention is the use of the nucleic acids produced and / or amplified according to the invention as nucleic acid probes.
  • the nucleic acids produced and / or amplified according to the invention can be used for diagnostic purposes in medicine and for research purposes.
  • kits for the detection of nucleic acid sequences and / or nucleic acids which contains at least one nucleic acid produced and / or amplified according to the invention.
  • the kit according to the invention can be used in the fields of biological dosimetry, tumor cytogenetics, microbiology and evolutionary biology and can be used here for the detection of genetic, carcinogenic or infectious diseases.
  • infectious diseases caused by retroviruses infectious diseases caused by retroviruses ("temperate phages")
  • a reaction mixture can be established with the host DNA and the nucleic acid sequence of the phages according to the present invention.
  • the specific nucleic acid of the phages acts as the starter molecule and the host DNA as the template.
  • the starter molecule of the phage nucleic acid can attach itself under suitable reaction conditions accumulate its complementary sequence in the host's DNA and assume its function as a starter.
  • the advantages here are (1) the detection of a phage infection and (2) knowledge of the mechanism of incorporation of the relevant phage nucleic acid into the host DNA, since the phage nucleic acid acts as a starter and accordingly the synthesized sequences ("elongation products") are partial sequences of the native host DNA.
  • new knowledge regarding the agents required for incorporation into the host DNA for example endonucleases or other DNA-cleaving and / or incorporating agents, can be obtained.
  • Figures 1 to 7 are schematic representations of preferred embodiments of the present invention, where (-) means any nucleotide, (*) means a nucleotide of the extension product and (
  • FIG. 8 is the photographic illustration of an agarose gel with amplification products produced by the method according to the invention. It means (from left to right): Lane 1: DNA sample specific for the centromer of human chromosome # 1 (pUC 1.77, Cooke et al., 1972); Lane 2: micro-dissected chromosome segment # 1; Lane 3: Nucleic acid sample obtained by microdisection specifically for the center of the human chromosome # 8. (amplification buffer No. 1 is used for lanes 1 to 3, the amount applied is 3 ⁇ l from a final volume of 50 ⁇ l after amplification has ended); Lane 4: DNA length standard marker No. III (Boehringer Mannheim, Mannheim, FRG) (amount applied 500 ng), • and lanes 5 to 8: as lanes 1 to 3, but in amplification buffer No. 2.
  • Lane 1 DNA sample specific for the centromer of human chromosome # 1 (pUC 1.77, Cooke et al., 1972); Lane 2: micro-diss
  • FIG. 9 is a photographic illustration of an agarose gel with ampli? fiction products. The following (from left to right) mean: lane 1: 500 ng DNA length standard marker No. III (Boehringer Mannheim, Mannheim, FRG) and lane 2. cDNA of the human myf3 gene (amount applied is 3 ⁇ l from a final volume of 50 ⁇ l after amplification has ended).
  • FIG. 10 is the photographic illustration of an agarose gel with amplification products produced by the method according to the invention.
  • the following from left to right: lane 1: cDNA of the human fibronectin gene (amount applied is 3 ⁇ l from a final volume of 50 ⁇ l after completion of the amplification) and lane 2: 500 ng DNA length standard marker No. III (Boehringer Mannheim, Mannheim, FRG).
  • FIG 11 is a photographic illustration of a "fluorescent multicolor" in situ hybridization using a modified method (Celeda et al., Z. Naturforsch. 47c (1992), 739-747) on human metaphase chromosomes.
  • Yellow hybridization markings (FITC) show the pUC 1.77 DNA sample specific for human chromosome # 1 from FIG. 8, lane 1; red hybridization markings (Texas Red) show the DNA sample from FIG. 8, lane 3, which is specific for human chromosome # 8.
  • FIG. 12 is a photographic illustration of a “fluorescent” in situ hybridization using the cDNA of the human myf3 gene from FIG. 9 according to a modified method (Celeda et al., Z. Naturforsch. 47c (1992), 739- 747) using a confocal laser scanning microscope. It means: Fig. 1: Hybridization to human rhabdomyosarcoma cells kept in culture; and Figure 2: Hybridizations to human lymphocytes obtained from peripheral blood.
  • DNA sample for human chromosome # 1 is added to a reaction solution (final volume 50 ⁇ l," amplification buffer No. 1 "), each containing 0.8 nmol of the nucleotides dATP, dCTP, dGTP and dTTP, 10 mmol tris (hydroxymethyl) aminomethane, 3 mmol MgCl 2 , 50 mmol KC1 and 5 units of a commercially available Taq polymerase.
  • the reaction mixture is introduced into a commercially available thermal cycler and 80 repetitions ("cycles") of the reaction sequences are carried out, with a further 5 units of Taq polymerase being added after 40 repetitions.
  • reaction conditions of a reaction sequence are (1) denaturing the nucleic acids contained in the reaction mixture at 90 ° C for 2 minutes and (2) synthesizing ("elongation") a new strand of nucleic acid using a supernatant strand as a template at 72 ° C for 3 minutes.
  • FIG. 8 lane 1 shows the result of this reaction by means of agarose gel electrophoresis.
  • reaction solution final volume 50 ⁇ l, “amplification buffer No. 2” each having 0.8 nmol of the nucleotides dATP, dCTP, dGTP and dTTP, 3 mmol MgCl 2 , 160 mmol NaCl, 0.02 vol .-% Tween 20 (polyoxyethylene sorbitan monolaurate) and 5 units of a commercially available Taq polymerase. 2. Amplification of a microdissected segment from Chromosom # 1
  • Example 2 The same reactions as in Example 1 are carried out, except that 6x10 ⁇ 9 g of a microdissected segment of chromosome # 1 is used.
  • Example 2 The same reactions as in Example 1 are carried out, except that 6 ⁇ 10 9 g of a microdissected nucleic acid sample specific for the centromere of human chromosome # 8 is used.
  • a myf-3 DNA sample 5 ⁇ 10 -9 g of a myf-3 DNA sample are added to a reaction solution (final volume 50 ⁇ l), each containing 0.8 nmol of the nucleotides dATP, dCTP, dGTP and dTTP, 3 mmol of MgCl 2 , 160 mmol of NaCl, 0 , 02% by volume of Tween 20 (polyoxyethylene sorbitan monolaurate) and 5 units of a commercially available Taq polymerase.
  • a reaction solution final volume 50 ⁇ l
  • Tween 20 polyoxyethylene sorbitan monolaurate
  • the reaction mixture is in a commercially available T h ermocycler introduced and 40 repeats ( "cycles") of the reaction sequences are carried out.
  • the reaction conditions of a reaction sequence are (1) denaturing the nucleic acids contained in the reaction mixture at 90 ° C. for 2 minutes and (2) synthesizing (“elongation”) of a new nucleic acid strand using an overhanging strand as a template at 72 ° C. for l minute.
  • FIG. 9, lane 2 shows the result of this reaction by means of agarose gel electrophoresis.
  • the reaction mixture is introduced into a commercially available thermal cycler and 80 repetitions ("cycles") of the reaction sequences are carried out, with a further 5 units of Taq polymerase being added after 40 repetitions.
  • the reaction conditions of a reaction sequence are (1) denaturing the nucleic acids contained in the reaction mixture at 94 ° C. for 2 minutes, (2) annealing at 54 ° C. for 2 minutes and (3) synthesizing (“elongation ”) a new strand of nucleic acid using a supernatant strand as a template at 72 ° C for 2 minutes.
  • FIG. 10, lane 1 shows the result of this reaction by means of agarose gel electrophoresis. 6.
  • FIGS. 11 and 12 The in situ hybridizations shown in FIGS. 11 and 12 are based on the method described by Celeda et al. (z. Naturforsch. 47c (1992), 739-747).

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Abstract

L'invention concerne un procédé de préparation et/ou d'amplification d'acides nucléiques, leur utilisation et un nécessaire contenant les acides nucléiques préparés selon l'invention. L'invention concerne notamment un procédé de préparation et/ou d'amplification de séquences d'acides nucléiques sans adjonction d'amorces.
PCT/DE1995/001003 1994-08-12 1995-07-28 Procede de preparation et d'amplification d'acides nucleiques WO1996005296A1 (fr)

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AU31604/95A AU3160495A (en) 1994-08-12 1995-07-28 Method of preparing and amplifying nucleic acids

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DEP4428651.1 1994-08-12
DE4428651A DE4428651C1 (de) 1994-08-12 1994-08-12 Verfahren zur Herstellung und Amplifikation von Nukleinsäuren

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997016546A1 (fr) * 1995-11-02 1997-05-09 Genencor International, Inc. Clonage moleculaire par multimerisation de plasmides
EP0812911A2 (fr) * 1996-06-10 1997-12-17 Japan Science and Technology Corporation Procédé pour la formation de polymère microgène macromoléculaire
WO1998010063A1 (fr) * 1996-09-03 1998-03-12 Protein Polymer Technologies, Inc. Procedes d'elaboration d'adn de synthese repetitif
US5834252A (en) * 1995-04-18 1998-11-10 Glaxo Group Limited End-complementary polymerase reaction
US5928905A (en) * 1995-04-18 1999-07-27 Glaxo Group Limited End-complementary polymerase reaction
WO2000041524A2 (fr) * 1999-01-11 2000-07-20 President And Fellows Of Harvard College Amplification isotherme d'adn
WO2001068674A2 (fr) * 2000-03-13 2001-09-20 Monsanto Technology Llc Proteines recombinantes contenant des unites recurrentes
WO2002030945A2 (fr) * 2000-10-13 2002-04-18 Domantis Limited Sequences d'acide nucleique enchainees

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19741714C2 (de) * 1997-09-22 2002-03-21 Inst Molekulare Biotechnologie Verfahren zur Synthese und Amplifikation von Nukleinsäuren
US6194179B1 (en) * 1999-07-20 2001-02-27 The Rockefeller University Method for preparing polynucleotide sequences and uses thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993009245A1 (fr) * 1991-10-31 1993-05-13 University Of Pittsburgh Hybridation inverse par buvardage en taches a l'aide de nomoneres tandem en tete-a-queue contenant des sondes synthesisees par des amorces complementaires decalees
WO1993009246A1 (fr) * 1991-11-01 1993-05-13 University Of Iowa Research Foundation Procede d'extraction d'une sequence adn de flanquement inconnue
WO1993019202A2 (fr) * 1992-03-10 1993-09-30 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Reaction a matrice echangeable
WO1993023572A1 (fr) * 1992-05-13 1993-11-25 University Of Texas Southwestern Medical Center At Dallas Therapie et diagnostic des etats pathologiques lies a la longueur du telomere et/ou a l'activite de la telomerase

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5215899A (en) * 1989-11-09 1993-06-01 Miles Inc. Nucleic acid amplification employing ligatable hairpin probe and transcription
NO904633L (no) * 1989-11-09 1991-05-10 Molecular Diagnostics Inc Amplifikasjon av nukleinsyrer ved transkriberbar haarnaalsprobe.
CA2077135A1 (fr) * 1991-08-30 1993-03-01 Joh-E Ikeda Methode d'amplification de l'adn
ZA929319B (en) * 1991-12-11 1993-05-24 Igen Inc Method for exponential amplification of nucleic acid by a single unpaired primer.

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993009245A1 (fr) * 1991-10-31 1993-05-13 University Of Pittsburgh Hybridation inverse par buvardage en taches a l'aide de nomoneres tandem en tete-a-queue contenant des sondes synthesisees par des amorces complementaires decalees
WO1993009246A1 (fr) * 1991-11-01 1993-05-13 University Of Iowa Research Foundation Procede d'extraction d'une sequence adn de flanquement inconnue
WO1993019202A2 (fr) * 1992-03-10 1993-09-30 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Reaction a matrice echangeable
WO1993023572A1 (fr) * 1992-05-13 1993-11-25 University Of Texas Southwestern Medical Center At Dallas Therapie et diagnostic des etats pathologiques lies a la longueur du telomere et/ou a l'activite de la telomerase

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
IJDO J W ET AL: "IMPROVED TELOMERE DETECTION USING A TELOMERE REPEAT PROBE (TTAGG)N GENERATED BY PCR", NUCLEIC ACIDS RESEARCH, vol. 19, no. 17, 11 September 1991 (1991-09-11), pages 4780, XP000227512 *
L.-M. HUANG AND K.-T. JEANG: "Long-range jumping of incompletely extended polymerase chain fragments generates unexpected products", BIOTECHNIQUES, vol. 16, no. 2, NATICK, MA, US, pages 242,244,246 *
W.A. RUDERT AND M. TRUCCO: "DNA polymers of protein binding sequences generated by PCR", NUCLEIC ACIDS RESEARCH, vol. 18, no. 21, 11 November 1990 (1990-11-11), IRL PRESS LIMITED,OXFORD,ENGLAND, pages 6460 *
WHITE M J ET AL: "CONCATEMER CHAIN REACTION: A TAQ DNA POLYMERASE-MEDIATED MECHANISM FOR GENERATING LONG TANDEMLY REPETITIVE DNA SEQUENCES", ANALYTICAL BIOCHEMISTRY, vol. 199, no. 2, 1 December 1991 (1991-12-01), pages 184 - 190, XP000236491 *

Cited By (16)

* Cited by examiner, † Cited by third party
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US5830713A (en) * 1986-11-04 1998-11-03 Protein Polymer Technologies, Inc. Methods for preparing synthetic repetitive DNA
US5928905A (en) * 1995-04-18 1999-07-27 Glaxo Group Limited End-complementary polymerase reaction
US6489146B2 (en) 1995-04-18 2002-12-03 Glaxo Group Limited End-complementary polymerase reaction
US5834252A (en) * 1995-04-18 1998-11-10 Glaxo Group Limited End-complementary polymerase reaction
WO1997016546A1 (fr) * 1995-11-02 1997-05-09 Genencor International, Inc. Clonage moleculaire par multimerisation de plasmides
EP0812911A3 (fr) * 1996-06-10 2001-04-18 Japan Science and Technology Corporation Procédé pour la formation de polymère microgène macromoleculaire
EP0812911A2 (fr) * 1996-06-10 1997-12-17 Japan Science and Technology Corporation Procédé pour la formation de polymère microgène macromoléculaire
WO1998010063A1 (fr) * 1996-09-03 1998-03-12 Protein Polymer Technologies, Inc. Procedes d'elaboration d'adn de synthese repetitif
WO2000041524A2 (fr) * 1999-01-11 2000-07-20 President And Fellows Of Harvard College Amplification isotherme d'adn
WO2000041524A3 (fr) * 1999-01-11 2001-02-08 Harvard College Amplification isotherme d'adn
US8709724B2 (en) 1999-01-11 2014-04-29 President And Fellows Of Harvard College Isothermal amplification of DNA
WO2001068674A2 (fr) * 2000-03-13 2001-09-20 Monsanto Technology Llc Proteines recombinantes contenant des unites recurrentes
WO2001068674A3 (fr) * 2000-03-13 2002-04-04 Monsanto Technology Llc Proteines recombinantes contenant des unites recurrentes
US7060467B2 (en) 2000-03-13 2006-06-13 Monsanto Technology Llc Recombinant proteins containing repeating units
WO2002030945A2 (fr) * 2000-10-13 2002-04-18 Domantis Limited Sequences d'acide nucleique enchainees
WO2002030945A3 (fr) * 2000-10-13 2002-07-18 Medical Res Council Sequences d'acide nucleique enchainees

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