WO1996005296A1 - Method of preparing and amplifying nucleic acids - Google Patents

Method of preparing and amplifying nucleic acids 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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WO
WIPO (PCT)
Prior art keywords
molecule
starter
nucleotide sequence
template
template molecule
Prior art date
Application number
PCT/DE1995/001003
Other languages
German (de)
French (fr)
Inventor
Karl-Otto Greulich
Dino Celeda
Original Assignee
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.)
Filing date
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/en

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Classifications

    • 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).

Abstract

The invention concerns a method for the preparation and/or amplification of nucleic acids, as well as the use of nucleic acids prepared in this way and a kit containing them. In particular, the invention concerns a method of preparing and/or amplifying nucleic-acid sequences without the addition of so-called primers.

Description

Verfahren zur Herstellung und Amplifikation von Nukleinsäuren Process for the production and amplification of nucleic acids
Diese Erfindung betrifft ein Verfahren zur Herstellung und/oder Amplifikation von Nukleinsäuren ohne zusätzliche Zugabe von definierten Startern ("Primern"), die Verwendung dieser Nukleinsäuren sowie einen die erfindungsgemäß herge¬ stellten Nukleinsäuren enthaltenden Kit.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.
Doppelsträngige Nukleinsäuresequenzen können durch Zugabe von für jeden der Stränge spezifischen Startern mit einer durch die Starter induzierten Verlängerungsreaktion unter Verwendung von Nukleotiden und einem für die Verlängerungsreaktion ge¬ eigneten Enzym amplifiziert werden. Diese als Polymerase-Ket¬ tenreaktion ("polymerase chain reaction", "PCR") bezeichnete Reaktion ist in EP-B1-0 201 184 beschrieben. Insbesondere ist bei der PCR ein molarer Überschuß der als Starter fungierenden definierten Oligonukleotide ("Primer") erforderlich, um die gewünschte Amplifikation der doppelsträngigen Nukleinsäurese¬ quenzen zu erhalten. Jeder durch einen der spezifischen Star¬ ter induzierte und enzymatisch synthetisierte Strang bildet im nächsten Reaktionszyklus die Matrize für einen durch den ande¬ ren Starter induzierten und enzymatisch zu synthetisierenden Strang, wobei die enzymatisch synthetisierten Stränge eines Zyklus jeweils zueinander komplementäre Nukleinsäuresequenzen sind. Die Reaktionszyklen können beliebig oft wiederholt wer¬ den, bis eine gewünschte Menge der doppelsträngigen Nuklein- säure im Reaktionsgemisch vorliegt. Nachteilig bei der PCR ist die zusätzliche Zugabe von Sequenz-spezifischen Oligonukleo- tid-Primern, was voraussetzt, daß sich genau zu diesen Oligo- nukleotid-Sequenzen entsprechende komplementäre Sequenzen in den zu amplifizierenden Nukleinsäuremolekülen befinden müssen. Dies kann u.a. zu keinem Amplifikationsprodukt führen, wenn keine entsprechenden komplementären Sequenzen in den zu ampli- fizierenden, doppelsträngig vorliegenden Nukleinsäuren vorhan¬ den sind. Ferner kann bei der PCR eine Amplifikation der Pri¬ mer durch Selbst-Anlagerung ( "Primer-Selfannealing" ) auftre¬ ten, was ein falsches Reaktionsprodukt sowie falsche Signale bei einer anschließenden in situ Hybridisierung mit den ampli- fizierten Nukleinsäuresequenzen zur Folge hat. Ferner wird im Reaktionsansatz für die PCR die Wahrscheinlichkeit einer Kon¬ tamination mit Fremd-Nukleinsäuren erhöht, da durch die zu¬ sätzliche Zugabe der Starter zwei weitere Pipetierschritte notwendig sind.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"), is described in EP-B1-0 201 184. In particular, a molar excess of the defined oligonucleotides ("primers") functioning as starters is required in order to obtain the desired amplification of the double-stranded nucleic acid sequences. In the next reaction cycle, 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. Among other things, this cannot lead to an amplification product if there are no corresponding complementary sequences in the amplified fictitious, double-stranded nucleic acids are present. Furthermore, amplification of the primer by self-attachment ("primer selfannealing") 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. Furthermore, 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.
Somit liegt der Erfindung die Aufgabe zugrunde, ein neues Ver¬ fahren zur Herstellung und/oder Amplifikation von Nukleinsäu¬ ren bereitzustellen, das keine Zugabe von spezifischen Oligo- nukleotid-Primern erfordert, um aus kleinsten Mengen von Nukleinsäuren, ohne den Zusatz von synthetisch hergestellter Fremd-DNS in Form von Primern, große Mengen an Nukleinsäuren herzustellen. Desweiteren sollen "Primer-Selfannealing" und somit ungewünschte amplifizierte Nukleinsäuresequenzen vermie¬ den und das Risiko einer Kontamination des Reaktionsansatzes mit Fremd-Nukleinsäuren minimiert werden.It is therefore the object of the invention to provide a new method for the production and / or amplification of nucleic acids which does not require the addition of specific oligonucleotide primers in order to make small amounts of nucleic acids without the addition of synthetically produced ones Foreign DNA in the form of primers to produce large amounts of nucleic acids. Furthermore, "primer selfannealing" and thus undesired amplified nucleic acid sequences are to be avoided and the risk of contamination of the reaction mixture with foreign nucleic acids is minimized.
Diese Aufgabe wird erfindungsgemäß durch ein Verfahren zur Herstellung und/oder Amplifikation von Nukleinsäuren gelöst, wobei der Reaktionsansatz ein einzelsträngig vorliegendes Nukleinsäuremolekül ("Startermolekül") mit einer als Starter wirkenden endständigen, vorzugsweise 3 ' -endständigen Nukleo- tidsequenz und ein einzelsträngig vorliegendes Nukleinsäuremo¬ lekül ("Matrizenmolekül") mit mindestens einer zur Anlagerung an die endständige Nukleotidsequenz eines Startermoleküls be¬ fähigten Nukleotidsequenz enthält, umfassend die SchritteThis 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
(a) Anlagern der endständigen im Startermolekül enthaltenen Nukleotidsequenz an eine im Matrizenmolekül enthaltenen Nukleotidsequenz unter Bildung eines überstehenden Stran¬ ges des Matrizenmoleküls,(a) attaching the terminal nucleotide sequence contained in the starter molecule to a nucleotide sequence contained in the template molecule to form a protruding strand of the template molecule,
(b) Synthetisieren eines Verlängerungsproduktes durch Induzie¬ ren mit der als Starter wirkenden endständigen Nukleotidsequenz des Startermoleküls unter Verwendung des überstehenden Stranges des Matrizenmoleküls als Matrize in Gegenwart von Nukleotiden und mindestens einem zur Syn¬ these des Verlängerungsproduktes geeigneten Agens, wobei eine das Verlängerungsprodukt enthaltende Nukleinsäure ("Reaktionsprodukt") erhalten wird,(b) Synthesizing an extension product by inducing the terminal one acting as a starter Nucleotide sequence of the starter molecule using the protruding strand of the template molecule as template in the presence of nucleotides and at least one agent suitable for the synthesis of the extension product, a nucleic acid containing the extension product (“reaction product”) being obtained,
(c) Trennen des Reaktionsproduktes vom Matrizenmolekül, und(c) separating the reaction product from the template molecule, and
(d) mindestens einmaliges Wiederholen der Schritte (a) bis(d) repeating steps (a) to at least once
(c) .(c).
Der Begriff "Nukleinsäuremolekül" bedeutet ein natives, halbsynthetisches, synthetisches oder modifiziertes Nuklein¬ säuremolekül aus Desoxyribonukleotiden und/oder Ribonukleoti- den und/oder modifizierten Nukleotiden, wie Aminonukleotiden oder [cu-S] -Triphosphatnukleotiden.The term “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.
Der Begriff "Startermolekül" bedeutet ein vorstehend definier¬ tes Nukleinsäuremolekül mit mindestens einer endständigen, vorzugsweise 3 ' -endständigen Nukleotidsequenz und einer die endständige Nukleotidsequenz flankierende Nukleotidsequenz, die vorzugsweise mindestens eine weitere zur Anlagerung an eine Nukleotidsequenz des Matrizenmoleküls befähigte Nukleo¬ tidsequenz enthält; vgl. Figuren l bis 7.The term "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.
In einer bevorzugten Ausfύhrungsform ist diese weitere Nukleo¬ tidsequenz am anderen Ende des Startermoleküls, vorzugsweise 5' -endständig, lokalisiert.In a preferred embodiment, this further nucleotide sequence is located at the other end of the starter molecule, preferably at the 5 'end.
Der Begriff "Matrizenmolekül" bedeutet ein vorstehend defi¬ niertes Nukleinsäuremolekül mit mindestens einer zur Anlage¬ rung an die endständige Nukleotidsequenz des Startermoleküls befähigten Nukleotidsequenz; vgl. Figuren 1 bis 7. In einer bevorzugten Ausführungsform enthält das Matrizenmole¬ kül mindestens eine endständige, vorzugsweise 3 ' -endständige Nukleotidsequenz, die zur Anlagerung an die endständige Nu¬ kleotidsequenz des Startermoleküls befähigt ist. Der Begriff "Reaktionsansatz" bedeutet ein Reaktionsgemisch, das neben den Nukleotiden und mindestens einem zur Synthese des Verlängerungsproduktes geeigneten Agens ein oder mehrere Startermoleküle und ein oder mehrere Matrizenmoleküle enthält, wobei weitere, nicht an dem erfindungsgemäßen Verfahren betei¬ ligte Nukleinsäuren vorhanden sein können. Die Startermoleküle und/oder die Matrizenmoleküle liegen in einer ausreichenden Konzentration, vorzugsweise mindestens etwa lxlO-15 g, im Re¬ aktionsansatz vor.The term “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. In a preferred embodiment, 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. The term “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.
Der Begriff "Anlagerung" bedeutet die Ausbildung von bei¬ spielsweise Wasserstoffbrücken zwischen einzelsträngigen, kom¬ plementären Bereichen von Nukleinsäuremolekülen, insbesondere zwischen den erfindungsgemäß definierten Nukleotidsequenzen der Startermoleküle und Matrizenmolekülen, bei einer geeigne¬ ten Temperatur, vorzugsweise 90 °C oder weniger, und gegebe¬ nenfalls bei einer geeigneten Salzkonzentration, vorzugsweise 50 bis 300 mM.The term “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.
Der Begriff "Verlängerungsprodukt" bedeutet eine an die end¬ ständige Nukleotidsequenz des Startermoleküls über beispiels¬ weise eine Phosphodiester-, Thioester- oder Amidbindung kova- lent gebundene, "synthetisierte" Nukleinsäureseuenz, deren Primärsequenz komplementär zu der entsprechenden Sequenz des Matrizenmoleküls ist.The term “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.
Der Begriff "ein zur Synthese des Verlängerungsproduktes ge¬ eignetes Agens" bedeutet ein natives Enzym oder ein synthe¬ tisch hergestelltes Agens, welches bei der Synthese des Ver¬ längerungsproduktes als Katalysator wirkt. Beispiele für na- tive Enzyme sind die Taq-Polymerase, das Klenow-Fragment der DNA-Polymerse I, die E. coli DNA-Polymerase I und die Reverse Transkriptase.The term "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. Examples of native enzymes are Taq polymerase, the Klenow fragment of DNA polymers I, E. coli DNA polymerase I and reverse transcriptase.
Der Begriff "Reaktionsprodukt" bedeutet eine, das Verlänge¬ rungsprodukt enthaltende Nukleinsäure, wobei das Reaktionspro- dukt per se bei jeder Wiederholung der Reaktionssequenz (a) bis (c) als Startermolekül und/oder als Matrizenmolekül gemäß den vorstehend aufgeführten Definitionen verwendet werden kann.The term “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.
In einer Ausführungsform der vorliegenden Erfindung sind das Startermolekül und das Matrizenmolekül gleich, wobei das Ma¬ trizenmolekül (bzw. das Startermolekül) mindestens eine Nukleotidsequenz enthält, die zur Anlagerung an die endstän¬ dige, vorzugsweise 3 ' -endständige Nukleotidsequenz des Star¬ termoleküls (bzw. des Matrizenmoleküls) befähigt ist; vgl. Fi¬ gur 3. Ferner kann das Matrizenmolekül (bzw. das Startermole¬ kül) eine weitere Nukleotidsequenz enthalten, die zur Anlage¬ rung der endständigen, vorzugsweise 3 ' -endständigen Nukleotid¬ sequenz des Verlängerungsproduktes befähigt ist. In einer bevorzugten Ausführungsform sind das Startermolekül und das Matrizenmolekül gleich und das Matrizenmolekül (bzw. das Startermolekül) enhält mindestens eine am anderen Ende, vorzugsweise 5' -endständig, lokalisierte Nukleotidsequenz, die zur Anlagerung an die endständige, vorzugsweise 3 ' -endständige Nukleotidsequenz des Startermoleküls (bzw. des Matrizenmole¬ küls) befähigt ist; vgl. Figur 4.In one embodiment of the present invention, 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. Furthermore, 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. In a preferred embodiment, 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.
In einer weiteren Ausführungsform der vorliegenden Erfindung enthält das Matrizenmolekül mindestens teilweise die komple¬ mentäre Sequenz des Startermoleküls, wobei das Matrizenmolekül mindestens eine, vorzugsweise zwei zur Anlagerung an die end¬ ständige Nukleotidsequenz des Startermoleküls befähigten Nukleotidsequenzen enthält; vgl. Figur 5. Vorzugsweise ist mindestens eine der zur Anlagerung an die endständige Nukleo¬ tidsequenz des Startermoleküls befähigten Nukleotidsequenzen des Matrizenmoleküls endständig, vorzugsweise 5' -endständig, lokalisiert.In a further embodiment of the present invention, 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. Preferably, 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.
In einer bevorzugten Ausführungsform der vorliegenden Erfin¬ dung ist das Matrizenmolekül die komplementäre Sequenz des StartermolekülT wobei mindestens zwei, vorzugsweise gleiche, zur Anlagerung an die endständige Nukleotidsequenz des Star- termoleküls befähigte Nukleotidsequenzen endεtändig lokali¬ siert sind; vgl. Figur 6.In a preferred embodiment of the present invention, 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.
In einer weiteren Ausführungsform der vorliegenden Erfindung ist das Startermolekül beispielsweise über eine Phosphodi- ester-, Thioester- oder Amidbindung kovalent an das Matrizen¬ molekül gebunden, so daß im Reaktionsansatz des erfindungsge¬ mäßen Verfahrens mindestens eine Nukleinsaure vorliegt, die die Primärsequenzen des Startermoleküls und des Matrizenmole¬ küls gemäß den vorstehend aufgeführten Definitionen enthält. Die endständige, vorzugsweise 3' -endständige Nukleotidsequenz dieser Nukleinsaure ist die zur Anlagerung an mindestens eine im Matrizenmolekül enthaltene Nukleotidsequenz befähigte Nukleotidsequenz des Startermoleküls; vgl. Figur 7. Die im Ma¬ trizenmolekül enthaltene Nukleotidsequenz kann beispielsweise 3'- oder 5' -endständig sein.In a further embodiment of the present invention, 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.
Die zur Anlagerung ("Hybridisierung") befähigten Nukleotidse¬ quenzen des Startermoleküls und/oder des Matrizenmoleküls sind vorzugsweise repetitive Sequenzen. Der Begriff "repetitive Se¬ quenzen" bedeutet sich wiederholende Sequenzen, wobei unter¬ schieden wird zwischen (1) repetitiven Genen, wie Gene von rRNA, tRNA, Histonen und Immunoglobulinen, (2) mittelrepetiti- ven Sequenzen, bestehend aus etwa 200 bis 300 Nukleotiden, und (3) hochrepetitive Sequenzen, bestehend aus kurzen Sequenzen von mindestens etwa 20 bp, die lOOOfach wiederholt sein können und sich, wie bei der "Alu-Familie" von Eukaryoten (Sequenzen von 300 bp) , über das ganze Genom verteilen.The nucleotide sequences of the starter molecule and / or of the template molecule which are capable of attachment ("hybridization") 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.
Ferner können die zur Anlagerung befähigten Nukleotidsequenzen des Startermoleküls und/oder des Matrizenmoleküls mindestens eine Erkennungssequenz für Endonukleasen oder für andere Nukleinsäure-spaltende Agentien, wie "molecular scissors", die auf einer Ausbildung von triple-Helix-DNS-Erkennungssequenzen basieren, enthalten.Furthermore, the 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.
Zur Markierung der erfindungsgemäß hergestellten Reaktionspro¬ dukte kann ein Teil der Nukleotide im Reaktionsansatz markiert sein. Geeignete Markierungen sind beispielsweise mit Biotin, Digoxigenin oder Fluoreszenzfarbstoffen gekoppelte Nukleotide oder mit einem radioaktiven Isotop markierte Nukleotide. Fer¬ ner können die Reaktionsprodukte selbst markiert werden, bei¬ spielsweise durch den Einbau von markierten Nukleotiden mit¬ tels "Nick-Translation" .To mark the reaction products produced according to the invention, 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. Furthermore, the reaction products themselves can be labeled, for example by incorporating labeled nucleotides using "nick translation".
In einer bevorzugten Ausführungsform der vorliegenden Erfin¬ dung ist der überstehende Strang des Matrizenmoleküls mit ei¬ ner genetischen, kanzerogenen oder infektiösen Krankheit ver¬ bunden. In diesem Fall handelt es sich um spezifische Nuklein¬ säuresequenzen, die modifiziert oder nativ bzw. direkt oder indirekt die Induktion einer solchen Krankheit verursachen.In a preferred embodiment of the present invention, the protruding strand of the template molecule is connected to a genetic, carcinogenic or infectious disease. In this case there are specific nucleic acid sequences which, modified or native or directly or indirectly, cause the induction of such a disease.
Das erfindungsgemäße Verfahren kann mit einzelsträngig oder doppelsträngig vorliegenden Nukleinsäuren durchgeführt werden, wobei doppelsträngig vorliegende Nukleinsäuren vor Schritt (a) in einzelsträngige Nukleinsäuren nach im Stand der Technik be¬ kannten Verfahren, wie Hitze-Denaturuierung oder pH-abhängige Denaturierung mit HC1 oder NaOH, überführt werden.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.
Der Reaktionsansatz hat ein geeignetes Volumen, beispielsweise 20 bis 200 μl , und enthält (l) eine für die Synthese des Ver¬ längerungsproduktes geeignete Konzentration an gewünschten Nukleotiden, vorzugsweise 5 bis 100 nmol, mehr bevorzugt etwa 20 nmol, (2) für die Synthese des Verlängerungsproduktes aus¬ reichende Einheiten eines synthetisierenden Agens, beispiels¬ weise l bis 15 Einheiten, vorzugsweise 5 Einheiten Taq-Polyme¬ rase, und (3) mindestens etwa lxlO"15 g Startermoleküle und Matrizenmoleküle bzw. Nukleinsäuren, die das Startermolekül und das Matrizenmolekül kovalent miteinander verbunden enthal¬ ten, in einer geeigneten Reaktionslόsung.The reaction mixture has a suitable volume, for example 20 to 200 μl, 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.
Die Reaktionslδsung enthält vorzugsweise MgCl2 (l bis 200 mmol, bevorzugt 1 bis 50 mmol, mehr bevorzugt 1 bis 20 mmol und am meisten bevorzugt 3 mmol), NaCl (30 bis 300 mmol, be¬ vorzugt 50 bis 250 mmol, mehr bevorzugt 100 bis 200 mmol und am meisten bevorzugt 160 mmol) und/oder KC1 (10 bis 70 mmol, bevorzugt 30 bis 70 mmol, mehr bevorzugt 40 bis 60 mmol und am meisten bevorzugt 50 mmol) und/oder Tris(hydroxymethyl)amino- methan (5 bis 50 mmol, bevorzugt 5 bis 30 mmol, mehr bevorzugt 5 bis 20 mmol und am meisten bevorzugt 10 mmol) und/oder Tween 20 (Polyoxyethylensorbitanmonolaurat) (0,01 bis 0,1 Vol.-%, bevorzugt 0,01 bis 0,06 Vol.-%, mehr bevorzugt 0,01 bis 0,04 Vol.-% und am meisten bevorzugt 0,02 Vol.-%) und gegebenen¬ falls Gelatine (0,1 bis 1 mmol). Der pH-Wert der Reaktionslδ- sung liegt in einem geeigneten, insbesondere von dem syntheti¬ sierenden Agens abhängigen Bereich.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). The pH of the reaction solution is in a suitable range, in particular depending on the synthesizing agent.
In Schritt (a) des erfindungsgemäßen Verfahrens lagern sich die endständigen Nukleotidsequenzen der Startermoleküle an in den Matrizenmolekülen enthaltene Nukleotidsequenzen unter Bil¬ dung überstehender Stränge, die als Matrize wirken, an. Diese Anlagerung wird bei im Reaktionsansatz zunächst doppel- strängig vorliegenden Nukleinsäuren und vor Schritt (a) dena¬ turierten und somit in Einzelstrangform überführten Nuklein¬ säuren auch als "versetzte Renaturierung" bezeichnet. Die Anlagerung erfolgt bei einer Temperatur, die jeweils von der Art der im Reaktionsansatz vorliegenden Nukleinsäuren ab¬ hängt.In 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. In the case of nucleic acids which are initially double-stranded in the reaction batch and nucleic acids denatured before step (a) and thus converted into single-strand form, 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.
Beispielsweise wird die Anlagerung der Nukleinsäuremoleküle, die von chromosomaler DNS mit mittelrepetitiven und hochrepe- titiven Sequenzen, beispielsweise "Alu-Sequenzen", stammen, bei einer Temperatur zwischen 70 und 90 °C durchgeführt. Dabei kann die Anlagerung derart erfolgen, daß gemäß Figur 7 die einzelsträngig vorliegende Nukleinsaure mit beispielsweise "inverted repeats" enthaltenden "Alu-Sequenzen" unter Bildung einer Schleife mit der endständigen, vorzugsweise 3' -endstän¬ digen Alu-Sequenz an eine in der Nukleinsaure enthaltene kom¬ plementäre "Alu-Sequenz" hybridisiert, wobei sich gemäß der vorstehend aufgeführten Definitionen die endständige "Alu-Se¬ quenz im Bereich des Startermoleküls und die komplementäre "Alu-Sequenz im Bereich des Matrizenmoleküls befinden. Somit kann eine endständige "Alu-Sequenz" in Schritt (b) des erfin¬ dungsgemäßen Verfahrens die Synthese eines Verlängerungspro- duktes durch beispielsweise die Taq-Polymerase induzieren, wo¬ bei der überstehende Strang der einzelsträngigen Nukleins ure als Matritze verwendet wird.For example, the attachment of the nucleic acid molecules which originate from chromosomal DNA with medium repetitive and highly repetitive sequences, for example “Alu sequences”, is carried out at a temperature between 70 and 90 ° C. In this case, 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. Thus, 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.
In beiden Fällen, nämlich "versetzte Renaturierung" oder Schleifenbildung bei chromosomaler DNS, ist die Temperatur der Anlagerung bzw. "Renaturierungstemperatur" von Bedeutung. Re¬ petitive Sequenzen renaturieren aufgrund ihres häufigen Vor¬ kommens und ihres zum Teil großen A-T Gehalts schneller. Hoch- repetitive DNS renaturiert schon bei Temperaturen unter 90 °C. Die Renaturierung ist aber zum großen Teil unspezifisch, wo¬ durch teilweise eine versetzte Anlagerung ("Hybridisierung") der einzelnen Stränge erreicht wird. Dies macht sich die vor¬ liegende Erfindung zum Nutzen. Die schnelle Renaturierung der einzelsträngig vorliegenden Stränge bzw. die schnelle Renatu¬ rierung einer einzelsträngig vorliegenden Nukleinsaure unter Schleifenbildung führt dazu, daß bei der erfindungsgemäßen Herstellung von Nukleinsäuren aus chromosomaler DNS der soge¬ nannte "Annealingschritt", welcher bei der PCR erforderlich ist, unnötig ist. Dies führt dazu, daß in den chromosomalen Nukleinsäuren enthaltene "single copy" Gene bedeutend lang¬ samer renaturieren und somit unter den gewählten Bedingungen von beispielsweise über 70 °C nicht oder nur viel langsamer renaturieren und damit keine Startsequenzen für eine Elonga- tion bilden können. Wenn beispielsweise eine hochrepetitive Nukleinsäuresequenz vor dem "Single copy" Gen in Synthese¬ bzw. Elongationsrichtung vorliegt, bietet sich die Mög¬ lichkeit, das entsprechende Gen vollständig, d.h. einschlie߬ lich seiner "Exons" (kodierende Sequenzen) und seiner "Introns" (nicht-kodierende Sequenzen) , herzustellen. Dies hat den Vorteil, daß in diesem Fall die genaue Sequenz des betref¬ fenden Gens in der nativen DNS erhalten wird und man somit nicht auf Kopien ("cDNA") der mRNS der betreffenden Gene ange¬ wiesen ist, die keine Introns mehr enthalten. Diese Möglich¬ keit ist für die Analyse von beispielsweise des menschlichen Genoms von großer Bedeutung, da sie die Herstellung von spezi- fischer DNS aus bestimmten Chromosomenregionen in Verbindung mit einer exakten Analyse des nativen Zustands erlaubt .In both cases, namely "staggered renaturation" or loop formation in chromosomal DNA, 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. If, for example, a highly repetitive nucleic acid sequence is present in front of the "single copy" gene in the direction of synthesis or elongation, there is the possibility of the corresponding gene completely, ie including its "exons" (coding sequences) and its "introns" ( non-coding sequences). This has the advantage that in this case the exact sequence of the gene in question is obtained in the native DNA and that there is therefore no need for copies ("cDNA") of the mRNA of the genes in question which no longer contain introns. This possibility is of great importance for the analysis of, for example, the human genome, since it involves the production of specific fischer DNA from certain chromosome regions in connection with an exact analysis of the native state allowed.
Bei der Amplifikation von doppelstrangig vorliegender cDNA (Menge im Reaktionsansatz vorzugsweise lxlO-9 bis lxlO"8 g, mehr bevorzugt 6xi0~9 g) , die "Single copy"-Sequenzen reprä¬ sentieren und somit keine hochrepetitiven Abschnitte in ihrer Sequenz aufweisen, wird die Anlagerung je nach Zusammensetzung der Nukleinsäuresequenz bei einer Temperatur von etwa 40 bis 80 °C durchgeführt. Damit wird einzelnen zum Teil endständigen AT-reichen bzw. GC-reichen Bereichen der Startermoleküle er¬ möglicht, an komplementäre Nukleotidsequenzen entweder inner¬ halb der einzelsträngigen Nukleinsäuren unter Schleifenbildung oder versetzt zu renaturieren. Dabei sollte der Anlagerungs- schritt zur Vermeidung einer vollständigen Renaturierung der zu amplifizierenden Nukleinsäuren eine Dauer von einer Stunde nicht überschreiten.In the amplification of double-stranded cDNA (amount in the reaction mixture preferably lxlO -9 to lxlO " 8 g, more preferably 6xi0 ~ 9 g), which represent" single copy "sequences and thus have no highly repetitive sections in their sequence depending on the composition of the nucleic acid sequence, 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.
Alle vorstehenden Ausführungen zu Schritt (a) des erfindungs- gemäßen Verfahrens sind mutatis mutandis auf die Ausführungs- form, bei der Startermolekül und Matrizenmolekül gleich sind, anwendbar.All of the above statements regarding 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.
In Schritt (b) wird die Synthese des Verlängerungsproduktes ("Elongation") bei einer Temperatur durchgeführt, die insbe¬ sondere von dem zur Synthese geeigneten Agens abhängt, wobei Reaktionsprodukte gemäß der vorstehend aufgeführten Definition erhalten werden. Beispielsweise erfolgt die Synthese bei Ver¬ wendung der Taq-Polymerase bei einer Temperatur zwischen 70 bis 80 °C, vorzugsweise 72 °C, für 1 bis 15 Minuten, vorzugs¬ weise 5 Minuten.In 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. For example, 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.
In Schritt (c) wird das Trennen des Reaktionsproduktes vom Ma¬ trizenmolekül beispielsweise durch Erhitzen des Reaktionsgemi¬ sches ("Denaturierung") auf 90 bis 100 °C, vorzugsweise 95 °C, für i bis 15 Minuten, vorzugsweise 5 Minuten, erreicht. Die Reaktionssequenz (a) bis (c) wird in Schritt (d) des er¬ findungsgemäßen Verfahrens mindestens einmal wiederholt. Ins¬ besondere wird das erfindungsgemäße Verfahren l bis 200 mal, vorzugsweise 80 mal wiederholt, wobei nach jeder 40. Wiederho¬ lung gegebenenfalls weitere Einheiten des für die Synthese ge¬ eigneten Agens, bespielsweise 1 bis 15 Einheiten, vorzugsweise 5 Einheiten Taq-Polymerase zugegeben werden.In 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. In particular, 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.
Die nach dem erfindungsgemäßen Verfahren hergestellten Reakti¬ onsprodukte können mit geeigneten Verfahren und/oder Mitteln physikalisch (z.B. durch Ultraschall) und/oder chemisch (z.B. durch "molecular scissors") und/oder enzymatisch (z.B. durch Endonukleasen) mindestens einmal gespalten werden.The 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.
Ein weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung der erfindungsgemäß hergestellten und/oder amplifi- zierten Nukleinsäuren als Nukleinsäuresonden. Insbesondere können die erfindungsgemäß hergestellten und/oder amplifizier- ten Nukleinsäuren zu diagnostischen Zwecken in der Medizin so¬ wie zu Forschungszwecken verwendet werden.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. In particular, the nucleic acids produced and / or amplified according to the invention can be used for diagnostic purposes in medicine and for research purposes.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Kit zum Nachweis von Nukleinsäuresequenzen und/oder von Nuklein¬ säuren, der mindestens eine erfindungsgemäß hergestellte und/oder amplifizierte Nukleinsaure enthält. Insbesondere kann der erfindungsgemäße Kit auf den Gebieten der biologischen Do- simetrie, der Tumorzytogenetik, der Mikrobiologie und der Evo¬ lutionsbiologie verwendet werden und hier zum Nachweis von ge¬ netischen, kanzerogenen oder infektiösen Krankheiten verwendet werden. Beispielsweise kann bei durch Retroviren ("temperente Phagen") verursachten infektiösen Krankheiten ein Reaktionsge¬ misch mit der Wirts-DNS und der Nukleinsäuresequenz der Phagen gemäß der vorliegenden Erfindung erstellt werden. Dabei fun¬ giert die spezifische Nukleinsaure der Phagen als Startermole¬ kül und die Wirts-DNS als Matrize. Wenn eine Phageninfektion in der Wirts-DNS vorliegt, kann sich das Startermolekül der Phagennukleinsäure unter geeigneten Reaktionsbedingungen an seine komplementäre Sequenz in der Wirts-DNS anlagern und seine Funktion als Starter übernehmen. Die Vorteile hierbei sind (l) der Nachweis einer Phageninfektion und (2) Erkennt¬ nisse über den Inkorporationsmechanismus der betreffenden Pha¬ gennukleinsäure in die Wirts-DNS, da die Phagennukleinsäure als Starter fungiert und demgemäß die synthetisierten Sequen¬ zen ("Elongationsprodukte") teilweise Sequenzen der nativen Wirts-DNS sind. Dadurch können neue Erkenntnisse bezüglich der für die Inkorporation in die Wirts-DNS benötigten Agenzien, beispielsweise Endonukleasen oder andere DNS-spaltende und/oder inkorporierende Agenzien, erhalten werden.Another object of the present invention is a kit 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. In particular, 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. For example, in the case of 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. If a phage infection is present in the host DNA, 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. As a result, 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.
Die Figuren zeigen:The figures show:
Figuren l bis 7 sind schematische Darstellungen bevorzugter Ausführungsformen der vorliegenden Erfindung, wobei (-) eine beliebiges Nukleotid bedeutet, (*) ein Nukleotid des Verlänge¬ rungsproduktes bedeutet und ( | ) komplementäre Nukleotide be¬ deuten.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 (|) mean complementary nucleotides.
Figur 8 ist die photographische Abbildung eines Agarosegels mit nach dem erfindungsgemäßen Verfahren hergestellten Ampli- fikationsprodukten. Es bedeuten (von links nach rechts) : Bahn 1: DNS-Probe spezifisch für das Centromer des menschlichen Chromosoms #1 (pUC 1.77, Cooke et al., 1972); Bahn 2: mikro- dissektiertes Chromosomensegment #1; Bahn 3: durch Mikrodis- sektion gewonnene Nukleinsäure-Probe spezifisch für das Cen¬ tromer des menschlichen Chromosoms #8. (für die Bahnen 1 bis 3 wird jeweils der Amplifikationspuffer Nr.l verwendet, die auf¬ getragene Menge beträgt jeweils 3 μl aus einem Endvolumen von 50 μl nach beendeter Amplifikation) ; Bahn 4 : DNS-Längen- standardmarker Nr. III (Boehringer Mannheim, Mannheim, FRG) (aufgetragene Menge 500 ng) ,• und Bahnen 5 bis 8: wie Bahnen l bis 3, jedoch in Amplifikationspuffer Nr. 2.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 μl from a final volume of 50 μl 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.
Figur 9 ist die photographische Abbildung eines Agarosegels mit nach dem erfindungsgemäßen Verfahren hergestellten Ampli- fikationsprodukten. Es bedeuten (von links nach rechts) : Bahn 1: 500 ng DNS- ängenstandardmarker Nr. III (Boehringer Mann¬ heim, Mannheim, FRG) und Bahn 2.- cDNA des menschlichen myf3- Gens (aufgetragene Menge beträgt 3 μl aus einem Endvolumen von 50 μl nach beendeter Amplifikation) .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 μl from a final volume of 50 μl after amplification has ended).
Figur 10 ist die photographische Abbildung eines Agarosegels mit nach dem erfindungsgemäßen Verfahren hergestellten Ampli- fikationsprodukten. Es bedeuten (von links nach rechts) : Bahn l: cDNA des menschlichen Fibronectin-Gens (aufgetragene Menge beträgt 3 μl aus einem Endvolumen von 50 μl nach beendeter Am¬ plifikation) und Bahn 2: 500 ng DNS-Längenstandardmarker Nr. III (Boehringer Mannheim, Mannheim, FRG) .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 μl from a final volume of 50 μl after completion of the amplification) and lane 2: 500 ng DNA length standard marker No. III (Boehringer Mannheim, Mannheim, FRG).
Figur 11 ist eine photographische Abbildung einer "fluorescent multicolor" in si tu Hybridisierung nach einem modifizierten Verfahren (Celeda et al., Z. Naturforsch. 47c (1992), 739-747) an menschlichen Metaphase-Chromosomen. Gelbe Hybridisierungs- markierungen (FITC) zeigen die für das menschliche Chromosom #1 spezifische pUC 1.77 DNS-Probe aus Fig. 8, Bahn l; rote Hy- bridisierungsmarkierungen (Texas Red) zeigen die für das men¬ schliche Chromosom #8 spezifische DNS-Probe aus Fig. 8, Bahn 3.Figure 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.
Figur 12 ist eine photographische Abbildung einer "fluores¬ zierenden" in si tu Hybridisierung unter Verwendung der cDNA des menschlichen myf3-Gens aus Fig. 9 nach einem modifizierten Verfahren (Celeda et al. , Z. Naturforsch. 47c (1992), 739-747) mittels eines konfokalen Laser-Scanning-Mikroskops. Es bedeu¬ ten: Bild l: Hybridisierung an menschliche, in Kultur gehal¬ tene Rhabdomyosarkomzellen; und Bild 2: Hybridisierungen an menschliche, aus peripherem Blut gewonnenen Lymphozyten.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.
Die nachstehenden Beispiele erläutern die Erfindung. 1. Amplifikation einer für das Centromer des menschlichen Chromosoms #1 spezifischen DNS-ProbeThe following examples illustrate the invention. 1. Amplification of a DNA sample specific for the centromere of human chromosome # 1
6xl0"9 g einer im Handel erhältlichen pUC 1.77 DNS-Probe für das menschliche Chromosom #1 wird zu einer Reaktionslδsung (Endvolumen 50 μl, "Amplifikationspuffer Nr. 1") zugegeben, die jeweils 0,8 nmol der Nukleotide dATP, dCTP, dGTP und dTTP, 10 mmol Tris (hydroxymethyl)aminomethan, 3 mmol MgCl2, 50 mmol KC1 und 5 Einheiten einer im Handel erhältlichen Taq-Polymerase enthält.6xl0 " 9 g of a commercially available pUC 1.77 DNA sample for human chromosome # 1 is added to a reaction solution (final volume 50 μl," 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.
Der Reaktionsansatz wird in einem im Handel erhältlichen Thermocycler eingebracht und 80 Wiederholungen ("Zyklen") der Reaktionssequenzen werden durchgeführt, wobei nach 40 Wiederholungen weitere 5 Einheiten der Taq-Polymerase zuge¬ geben werden.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.
Die Reaktionsbedingungen einer Reaktionssequenz sind (l) Denaturieren der im Reaktionsgemisch enthaltenen Nuklein¬ säuren bei 90 °C für 2 Minuten und (2) Synthetisieren ("Elongation") eines neuen Nukleinsäurestranges unter Ver¬ wendung eines überstehenden Stranges als Matrize bei 72 °C für 3 Minuten.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") a new strand of nucleic acid using a supernatant strand as a template at 72 ° C for 3 minutes.
In Figur 8, Bahn 1, ist das Ergebnis dieser Reaktion mit¬ tels Agarose-Gelelektrophorese dargestellt.FIG. 8, lane 1, shows the result of this reaction by means of agarose gel electrophoresis.
In Bahn 5 von Figur 8 ist ein Ergebnis mit der gleichen DNS-Probe dargestellt, wobei hier die Reaktionslδsung (Endvolumen 50 μl, "Amplifikationspuffer Nr. 2") jeweils 0,8 nmol der Nukleotide dATP, dCTP, dGTP und dTTP, 3 mmol MgCl2, 160 mmol NaCl, 0,02 Vol.-% Tween 20 (Polyoxyethylen- sorbitanmonolaurat) und 5 Einheiten einer im Handel erhält¬ lichen Taq-Polymerase enthält. 2. Amplifikation eines mikrodissektierten Segments vom Chromo¬ som #1A result with the same DNA sample is shown in lane 5 of FIG. 8, the reaction solution (final volume 50 μl, “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
Die gleichen, wie in Beispiel l aufgeführten Reaktionen werden durchgeführt, mit der Ausnahme, daß 6x10~9 g eines mikrodissektierten Segments von Chromosom #1 verwendet wird.The same reactions as in Example 1 are carried out, except that 6x10 ~ 9 g of a microdissected segment of chromosome # 1 is used.
Die Ergebnisse sind in Figur 8, Bahnen 2 (Amplifikations- puffer Nr. 1) und 6 (Amplifikationspuffer Nr. 2), darge¬ stellt.The results are shown in FIG. 8, lanes 2 (amplification buffer No. 1) and 6 (amplification buffer No. 2).
3. Amplifikation einer mikrodissektierten. für das Centromer des menschlichen Chromosoms #8 spezifischen Nukleinsäure- Probe3. Amplification of a microdissected. for the centromer of human chromosome # 8 specific nucleic acid sample
Die gleichen, wie in Beispiel l aufgeführten Reaktionen werden durchgeführt, mit der Ausnahme, daß 6xl0"9 g einer mikrodissektierten, für das Centromer des menschlichen Chromosoms #8 spezifischen Nukleinsaure-Probe verwendet wird.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.
Die Ergebnisse sind in Figur 8, Bahnen 3 (Amplifikations- puffer Nr. l) und 7 (Amplifikationspuffer Nr. .2) , darge¬ stellt.The results are shown in FIG. 8, lanes 3 (amplification buffer No. 1) and 7 (amplification buffer No. 2).
4. Amplifikation einer cDNA des menschlichen myf3-Gens4. Amplification of a cDNA of the human myf3 gene
5xl0-9 g einer myf-3 DNS-Probe werden zu einer Reaktionslδ¬ sung (Endvolumen 50 μl) zugegeben, die jeweils 0,8 nmol der Nukleotide dATP, dCTP, dGTP und dTTP, 3 mmol MgCl2, 160 mmol NaCl, 0,02 Vol.-% Tween 20 (Polyoxyethylensorbitan- monolaurat) und 5 Einheiten einer im Handel erhältlichen Taq-Polymerase enthält.5 × 10 -9 g of a myf-3 DNA sample are added to a reaction solution (final volume 50 μl), 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.
Der Reaktionsansatz wird in einem im Handel erhältlichen Thermocycler eingebracht und 40 Wiederholungen ("Zyklen") der Reaktionssequenzen werden durchgeführt. Die Reaktions- bedingungen einer Reaktionssequenz sind (1) Denaturieren der im Reaktionsgemisch enthaltenen Nukleinsäuren bei 90 °C für 2 Minuten und (2) Synthetisieren ("Elongation") eines neuen Nukleinsäurestranges unter Verwendung eines überste¬ henden Stranges als Matrize bei 72 °C für l Minute.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.
In Figur 9, Bahn 2, ist das Ergebnis dieser Reaktion mit¬ tels Agarose-Gelelektrophorese dargestellt.FIG. 9, lane 2, shows the result of this reaction by means of agarose gel electrophoresis.
5. Amplifikation einer cDNA-Nukleinsäuresonde des menschlichen Fibronectin-Gens5. Amplification of a cDNA nucleic acid probe of the human fibronectin gene
5xl0"9 g einer Fibronectin-DNS-Probe werden zu einer Reak¬ tionslδsung (Endvolumen 50 μl) zugegeben, die jeweils 0,8 nmol der Nukleotide dATP, dCTP, dGTP und dTTP, 10 mmol Tris(hydroxymethyl)aminomethan, 3 mmol MgCl , 50 mmol KC1 und 5 Einheiten einer im Handel erhältlichen Taq-Polymerase enthält.5 × 10 " 9 g of a fibronectin DNA sample are added to a reaction solution (final volume 50 μl), each containing 0.8 nmol of the nucleotides dATP, dCTP, dGTP and dTTP, 10 mmol of tris (hydroxymethyl) aminomethane, 3 mmol of MgCl , 50 mmol KC1 and 5 units of a commercially available Taq polymerase.
Der Reaktionsansatz wird in einem im Handel erhältlichen Thermocycler eingebracht und 80 Wiederholungen ("Zyklen") der Reaktionssequenzen werden durchgeführt, wobei nach 40 Wiederholungen weitere 5 Einheiten der Taq-Polymerase zuge¬ geben werden.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.
Die Reaktionsbedingungen einer Reaktionssequenz sind (l) Denaturieren der im Reaktionsgemisch enthaltenen Nuklein¬ säuren bei 94 °C für 2 Minuten, (2) Anlagerung ("Annealing") bei 54 °C für 2 Minuten und (3) Synthetisie¬ ren ("Elongation") eines neuen Nukleinsäurestranges unter Verwendung eines überstehenden Stranges als Matrize bei 72 °C für 2 Minuten.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.
In Figur 10, Bahn l, ist das Ergebnis dieser Reaktion mit¬ tels Agarose-Gelelektrophorese dargestellt. 6. In si tu HybridisierungenFIG. 10, lane 1, shows the result of this reaction by means of agarose gel electrophoresis. 6. In si tu hybridizations
Die in den Figuren 11 und 12 dargestellten in si tu Hybridi¬ sierungen werden nach dem von Celeda et al. (z. Natur- forsch. 47c (1992) , 739-747) beschriebenen Verfahren durch¬ geführt. 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).

Claims

PATENTANSPRÜCHE PATENT CLAIMS
1. Verfahren zur Herstellung und/oder Amplifikation von Nukleinsäuren, wobei der Reaktionsansatz ein einzelsträngiges Nukleinsäuremolekül ("Startermolekül") mit einer als Starter wirkenden endständigen Nukleotidsequenz und ein einzelsträngiges Nukleinsäuremolekül ("Matrizenmolekül") mit mindestens einer zur Anlagerung an die endständige Nukleotidsequenz eines Startermoleküls befähigten Nukleotidsequenz enthält, umfassend die Schritte1. A method for producing and / or amplifying nucleic acids, the reaction mixture comprising a single-stranded nucleic acid molecule ("starter molecule") with a terminal nucleotide sequence acting as a starter and a single-stranded nucleic acid molecule ("template molecule") with at least one for attachment to the terminal nucleotide sequence of a starter molecule contains qualified nucleotide sequence, comprising the steps
(a) Anlagern der endständigen im Startermolekül enthaltenen Nukleotidsequenz an eine im Matrizenmolekül enthaltenen Nukleotidsequenz unter Bildung eines überstehenden Stranges des Matrizenmoleküls,(a) attaching the terminal nucleotide sequence contained in the starter molecule to a nucleotide sequence contained in the template molecule to form a protruding strand of the template molecule,
(b) Synthetisieren eines Verlängerungsproduktes durch Induzieren mit der als Starter wirkenden endständigen Nukleotidsequenz des Startermoleküls unter Verwendung des überstehenden Stranges des Matrizenmoleküls als Matrize in Gegenwart von Nukleotiden und mindestens einem zur Synthese des Verlängerungsproduktes geeigneten Agens, wobei eine das Verlängerungsprodukt enthaltende Nukleinsaure ("Reaktionsprodukt*!) erhalten wird,(b) Synthesis of an extension product by induction with the terminal nucleotide sequence of the starter molecule acting as a starter using the protruding strand of the template molecule as template in the presence of nucleotides and at least one agent suitable for the synthesis of the extension product, wherein a nucleic acid containing the extension product ("reaction product * !) is obtained
(c) Trennen des Reaktionsprodukteε vom Matrizenmolekül, und(c) separating the reaction product from the template molecule, and
(d) mindestens einmaliges Wiederholen der Schritte (a) bis(d) repeating steps (a) to at least once
(c) .(c).
2. Verfahren nach Anspruch 1, wobei das Matrizenmolekül die komplementäre Sequenz des Startermoleküls ist .2. The method of claim 1, wherein the template molecule is the complementary sequence of the starter molecule.
3. Verfahren nach Anspruch l, wobei das Startermolekül und das Matrizenmolekül gleich sind. 3. The method according to claim 1, wherein the starter molecule and the template molecule are the same.
4. Verfahren nach Anspruch 1, wobei das Startermolekül kovalent an das Matrizenmolekül gebunden ist.4. The method of claim 1, wherein the starter molecule is covalently bound to the template molecule.
5. Verfahren nach Anspruch 3 oder 4, wobei das Reaktionsprodukt eine endständige Nukleotidsequenz enthält, die zur Anlagerung an mindestens einer Nukleotidsequenz des Matrizenmoleküls befähigt ist.5. The method according to claim 3 or 4, wherein the reaction product contains a terminal nucleotide sequence which is capable of attachment to at least one nucleotide sequence of the template molecule.
6. Verfahren nach einem der Ansprüche l bis 5, wobei mindestens eine zur Anlagerung an die endständige Nukleotidsequenz des Startermoleküls befähigte Nukleotidsequenz des Matrizenmoleküls endständig ist und im Schritt (b) das Matrizenmolekül als Startermolekül und das Startermolekül als Matrizenmolekül gemäß den Definitionen in Anspruch l verwendet wird.6. The method according to any one of claims 1 to 5, wherein at least one nucleotide sequence of the template molecule capable of attachment to the terminal nucleotide sequence of the starter molecule is terminal and used in step (b) the template molecule as the starter molecule and the starter molecule as the template molecule as defined in claim 1 becomes.
7. Verfahren nach einem der Ansprüche l bis 6, wobei die zur Anlagerung befähigten Nukleotidsequenzen im Startermolekül und/oder Matrizenmolekül repetitive Sequenzen sind.7. The method according to any one of claims 1 to 6, wherein the nucleotide sequences capable of attachment in the starter molecule and / or template molecule are repetitive sequences.
8. Verfahren nach einem der Ansprüche 1 bis 7, wobei das Startermolekül und/oder das Matrizenmolekül aus chromosomaler DNS stammt.8. The method according to any one of claims 1 to 7, wherein the starter molecule and / or the template molecule is derived from chromosomal DNA.
9. Verfahren nach einem der Ansprüche 1 bis 8, wobei der überstehende Strang des Matrizenmoleküls mit einer genetischen, kanzerogenen oder infektiösen Krankheit verbunden ist.9. The method according to any one of claims 1 to 8, wherein the protruding strand of the template molecule is associated with a genetic, carcinogenic or infectious disease.
10. Verfahren nach einem der Ansprüche l bis 9, wobei die zur Anlagerung befähigten Nukleotidsequenzen des Startermoleküls und/oder des Matrizenmoleküls mindestens eine Erkennungssequenz für Endonukleasen enthalten.10. The method according to any one of claims 1 to 9, wherein the nucleotide sequences of the starter molecule and / or the template molecule capable of attachment contain at least one recognition sequence for endonucleases.
11. Verfahren nach einem der Ansprüche 1 bis 10, wobei zusätzlich das Reaktionsprodukt jeder Reaktionssequenz (a) bis (c) als Startermolekül und/oder als Matrizenmolekül gemäß den Definitionen in Anspruch 1 verwendet wird.11. The method according to any one of claims 1 to 10, wherein additionally the reaction product of each reaction sequence (a) to (c) is used as a starter molecule and / or as a template molecule as defined in claim 1.
12. Verfahren nach einem der Ansprüche 1 bis 11, wobei das Agens T4-DNA-Polymerase, E. coli DNA-Polymerase I, Reverse Transkriptase oder ein synthetisierendes und/oder Hitze¬ stabiles Enzym ist.12. The method according to any one of claims 1 to 11, wherein the agent is T4 DNA polymerase, E. coli DNA polymerase I, reverse transcriptase or a synthesizing and / or heat-stable enzyme.
13. Verfahren nach einem der Ansprüche 1 bis 12, wobei mindestens ein Teil der Nukleotide eine Markierung aufweist.13. The method according to any one of claims 1 to 12, wherein at least some of the nucleotides have a label.
14. Verfahren nach einem der Ansprüche l bis 13, wobei die Reaktionsprodukte markiert werden.14. The method according to any one of claims 1 to 13, wherein the reaction products are labeled.
15. Verfahren nacch einem der Ansprüche 1 bis 14, wobei das Reaktionsprodukt mindestens einmal gespalten wird.15. The method according to any one of claims 1 to 14, wherein the reaction product is cleaved at least once.
16. Verfahren nach Anspruch 15, wobei das Reaktionsprodukt physikalisch und/oder chemisch und/oder enzymatisch gespalten wird.16. The method according to claim 15, wherein the reaction product is physically and / or chemically and / or enzymatically cleaved.
17. Verwendung der nach einem der Ansprüche 1 bis 16 hergestellten Reaktionsprodukte als Nukleinsäuresonden.17. Use of the reaction products prepared according to one of claims 1 to 16 as nucleic acid probes.
18. Diagnostischer Kit zum Nachweis von spezifischen in Nukleinsäuren enthaltenen Sequenzen und/oder von Nukleinsäuren, enhaltend die nach einem der Ansprüche 1 bis 16 hergestellten Reaktionsprodukte. 18. Diagnostic kit for the detection of specific sequences contained in nucleic acids and / or of nucleic acids containing the reaction products produced according to one of claims 1 to 16.
PCT/DE1995/001003 1994-08-12 1995-07-28 Method of preparing and amplifying nucleic acids WO1996005296A1 (en)

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