EP1476579A2 - Procede pour la reduction de la contamination d'arriere-plan apres des reactions de marquage - Google Patents

Procede pour la reduction de la contamination d'arriere-plan apres des reactions de marquage

Info

Publication number
EP1476579A2
EP1476579A2 EP03739498A EP03739498A EP1476579A2 EP 1476579 A2 EP1476579 A2 EP 1476579A2 EP 03739498 A EP03739498 A EP 03739498A EP 03739498 A EP03739498 A EP 03739498A EP 1476579 A2 EP1476579 A2 EP 1476579A2
Authority
EP
European Patent Office
Prior art keywords
marker
labeled
labeling
enzyme
luminescence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP03739498A
Other languages
German (de)
English (en)
Inventor
Christian Korfhage
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qiagen GmbH
Original Assignee
Qiagen GmbH
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 Qiagen GmbH filed Critical Qiagen GmbH
Publication of EP1476579A2 publication Critical patent/EP1476579A2/fr
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Definitions

  • the invention relates to a method for reducing the background contamination when carrying out labeling reactions on biomolecules, preferably on biopolymers such as peptides, proteins or nucleic acids with labeling substances.
  • Nucleic acids are labeled with modified nucleotides.
  • labeled substances are used as a code or sensor to identify other molecules or to track processes.
  • nucleic acids (DNA, RNA) of various lengths can be labeled via the enzymatic incorporation of labeled nucleotides.
  • probes can be produced which have a high marking density and a high sensitivity.
  • the object of the present invention is therefore to provide a method which makes it possible to discriminate similar properties of the labeled reactants. It is therefore the aim of the present invention to provide a substance preparation with as little background contamination as possible and easy to carry out.
  • nucleic acid fragments, oligonucleotides, proteins and peptides are labeled, for example, with radioactive compounds or with dyes.
  • the respective method for labeling is determined, for example, by the length of the nucleic acid fragments, the experimental requirements and the sensitivity of detection.
  • the labeling reaction using polymerases such as e.g. DNA or RNA polymerases or reverse transcriptases are catalyzed.
  • the exchange position is either the a or ⁇ position of the phosphate residues in 2'-deoxyribo, 3'-deoxyribo or 2'-ribonucleotides.
  • the 25 l mark is made at the C-5 position of the cytosine.
  • the introduction of the modification group changes the chemical structure of the labeled nucleotides. This has the consequence, for example, that the reaction conditions for the enzymatic incorporation have to be adapted to the changed substrate properties.
  • the membrane surface must be saturated with blot hybridizations and the cell or tissue surface in s / Yt / hybridizations with suitable blocking reagents.
  • Labeled nucleic acids are used in molecular biological cloning as a reagent or as a sample.
  • the labeling reagent can be covalently bound to the substance to be labeled or non-covalently associated with it.
  • labeled fragments of cloned DNA and / or oligonucleotides of a defined size are used as reagents for chemical and enzymatic sequencing, for nuclease S1 analysis of RNA and in so-called band-shift experiments.
  • labeled nucleic acid samples are required in hybridization techniques for the localization and binding of DNA and RNA of complementary sequences. These techniques include colony and PIaque hybridization, Southern and Northern analyzes, in s / Yu hybridizations and sequencing by hybridizations.
  • the success of introducing the label in DNA or RNA depends on the method used, e.g. End marking, "random priming", nick translation, in wrro transcription and variations of the polymerase chain reaction (PCR) etc.
  • [ ⁇ - 32 P] ATP is used for radioactive labeling of single-stranded hybridization samples or DNA for sequencing according to Maxam-Gilbert.
  • the ⁇ -phosphate group of the ATP is transferred by the enzyme T4 polynucleotide kinase to the 5'-terminal hydroxyl group of the oligodeoxynucleoside triphosphate or the DNA.
  • DNA oligonucleotides can be labeled enzymatically via the terminal transferase by attaching labeled dNTPs independently of the template (tailing).
  • DNA with 5 'protruding ends can be radiolabelled by one filling reaction of the Klenow fragment of E.coli DNA polymerase I at one or both 3' ends.
  • the [ ⁇ -32P] deoxynucleoside triphosphates are used, which are complementary to the respective first base of the 5 'single-strand ends.
  • nick translation also means that
  • E.coli DNA polymerase holoenzyme used. The reason for this is that in addition to polymerase activity, this method also requires 5 ' ⁇ 3' exonuclease activity.
  • the DNase catalyzes the formation of single strand breaks (nicks). To ensure that no further cleavages take place, a precisely set, low DNase concentration is required. The the
  • Single strand break flanking 3 'and 5' ends act as a substrate for the 5'- »3'-polymerase activity and the 5'- 3'-exonuclease activity.
  • the 5'- »3'-exonuclease activity is responsible for the successive degradation of the 5'-phosphorylated nucleotides, while synchronously due to the 5'- 3'-polymerase activity resulting gaps are filled with new, labeled nucleotides.
  • the primers represent a mixture of all possible hexanucleotides (random primers), so that - statistically speaking - every target sequence is covered and hybridization can take place at any desired location.
  • the Klenow fragment the large subtilisin fragment of the DNA polymerase holoenzyme, extends the primers in a template-dependent reaction. Unlabeled dNTPs and hapten-modified dUTPs are incorporated into the elongation reaction. Since the template strands are replicated, a new synthesis takes place. In this way, high probe yields with over 100% of the template DNA used can be obtained via strand displacement.
  • Digoxigenin-labeled probes which are generated by the "random priming" method, can be used to achieve high detection sensitivities in the sub-picogram range
  • Labeled RNA probes of high specific activity can be produced by in vitro transcription of cloned DNA fragments. Suitable promoters are required for this, e.g. in cloning vectors
  • Transcription vectors such as vectors from the Ribo Gemini series (pGEM-3 or pGEM-4). These vectors can be used to produce RNA samples of opposite orientation (eg "sense” and “antisense”) in the case of the corresponding RNA polymerase and in trans-transcription reactions. To do this, the vector are linearized downstream of the sequence to be amplified so that no RNA fragments are generated which run around the entire vector (“run-around” transcripts). In this way, only the desired cloned sequence is labeled with [ ⁇ - 32 P] nucleotides (ATP or CTP).
  • ATP ⁇ - 32 P] nucleotides
  • the first non-radioactive methods were developed as early as 1980 and are based on the labeling of nucleic acid samples with dinitrophenol, bromodeoxyuridine and biotin.
  • biotinylated samples are detected via an interaction with streptavidin, which is often conjugated with alkaline phosphatase as a reporter enzyme, via the enzymatic activity of the phosphatase.
  • the homogeneous DNA labeling is carried out either by "random priming" with the large fragment of E. coli DNA polymerase I (Klenow enzyme), nick translation E. coli DNA polymerase I (Kornberg enzyme) or by PCR amplification Taq polymerase reached.
  • the label densities are one label per 25 to 36 base pairs.
  • Oligonucleotides can be labeled enzymatically using the terminal transferase reaction; depending on the substrate, one to five labels per oligonucleotide are attached here.
  • Nucleic acids can be labeled with biotin and digoxigenin (DIG), which are linked via a nitrophenylazido group. Irradiation of the nitrophenylazido group with UV light leads to a photochemical reaction. Here, reactive nitrenes are split off. Detection of non-radioactive labeled samples after hybridization
  • DIG digoxigenin
  • Chemiluminescence is a fast and sensitive parameter for the detection of DNA.
  • Antibodies are used for this purpose which specifically target the markings made in the DNA, e.g. Bind biotin, fluorescein or digoxigenin, and are coupled, for example, to horseradish peroxidase (HRP) or alkaline phosphatase. Both enzymes can be used in reactions in which light is emitted or a color conversion takes place.
  • HRP horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • Fluorescence labeling with fluorophores is mainly used for polypeptides or small proteins, especially for those that cannot be detected with Coomassie blue and / or with a silver stain.
  • the fluorescent label can be used as an alternative to staining techniques.
  • Fluorophore labeling is widely used in nucleic acids in hybridization systems such as in microarrays.
  • the object of the present invention is to provide a method in which the background contamination is reduced after the marking reaction has taken place.
  • non-radioactive markers can also be used according to the invention.
  • reporter groups are known from the prior art [C. Kessler, Nonradioactive Analysis of Biomolecules, J. Biotechnol. 35 (1994) 165]; among these, the following are preferred:
  • Fluorescence markers - such as markers for direct fluorescence: fluorescein (FITC, FLUOS), cyanines, Alexa fluorophores, rhodamine (RHODOS, RESOS, RESIAC), hydroxycoumarin (AMCA), benzofuran, Texas red, biman, ethidium / Tb 3+ ,
  • Fluorescence markers for time-triggered fluorescence such as lanthanoid (Eu 3+ / Tb 3+ ) complexes, micelles or chelates.
  • Fluorescence markers for fluorescence energy transfer such as fluorescein: rhodamine.
  • Luminescent markers for chemiluminescence e.g. (Iso) luminol derivatives or acridine esters.
  • Luminescent markers for electroluminescence such as Ru 2+ - (2,2'-bipyridyl) 3 - complexes.
  • Luminescence markers for luminescence energy transfer e.g. Rhodamine: luminol.
  • Metal markers e.g. metal-marked - especially Au- and Ag-marked
  • Enzyme markers for direct enzyme coupling e.g. Alkaline phosphatase (AP)
  • Oxidase glucose-6-phosphate dehydrogenase, hexokinase, bacterial luciferase,
  • Enzyme markers for enzyme substrate transfer e.g. Glucose oxidase: horseradish
  • Enzyme markers for enzyme complementation e.g. Inactive? -Galactosidase: ⁇ - peptide.
  • Polymeric markers e.g. Latex dye particles or polyethyleneimine.
  • the labeling agent can be covalently bound to the substance to be labeled (nucleic acids, peptides, oligopeptides, proteins or other biopolymers) or can be non-covalently associated with it.
  • the task is to avoid a high background signal, which leads to a short range of sensitivity and dynamic range of the measurements.
  • this object is achieved by a method in which, before the purification of the labeled substance, preferably before the end of the labeling reaction, particularly preferably in the last third and very particularly preferably at the end of the labeling reaction, an unlabeled substance, preferably an unlabeled one Adds derivative of the starting material used and particularly preferably the corresponding unlabelled starting material to the reaction mixture.
  • the unlabeled substance is preferably chemically, physically or structurally related to the substance used for the marking or a derivative thereof, or particularly preferably with the starting material except for the actual marking.
  • the concentration ratio of the unlabelled reagent to the labeled reagent is preferred in an interval from 1: 1 to 1000: 1, particularly preferably in an interval from 10: 1 to 100: 1.
  • Example 1 shows the reduction in noise after the addition of various amounts of unlabeled reagent after the end of the labeling reaction described in Example 1.
  • Example 2 shows the signal to noise ratio after the addition of various amounts of unlabeled reagent after the end of the labeling reaction described in Example 2.
  • 3 graphically represents the reduction in noise after the addition of unlabeled reagent after the end of the labeling reaction described in Example 3.
  • nucleic acid solutions are purified using a silica purification step (for example "QiaQuick" from Qiagen, D-40724 Hilden).
  • the RNA binds to the silica membrane during the purification process.
  • the eluate obtained in this way which should not contain any free radiolabelled nucleotides, but purified RNA, is measured.
  • a continuous reduction of the background contamination after addition of a 1.7 mM dNTP, 5 mM dNTP to a 10 mM dNTP solution is detected.
  • reaction batches contain Omniscript Reverse Transcriptase (from Qiagen, D-40724 Hilden), while the other reaction batches contain no enzyme for the incorporation of radioactively labeled nucleotides and thus serve as background control.
  • These mixtures are incubated for 1 h at 37 ° C. and then supplemented with 10 ⁇ l of a mixture which contains unlabelled nucleotides of different concentrations in different reaction batches. 10 ⁇ l of water (0 mM dNTP) were added to a reaction mixture. This serves as a control approach.
  • the nucleic acid solutions are purified using a silica purification step (for example "QiaQuick" from Qiagen, D-40724 Hilden).
  • the RNA and radioactively labeled cDNA bind to the silica membrane during the purification process (commercially available from Qiagen, D-40724 Hilden).
  • the RNA binds to the silica membrane.
  • the eluate which should not contain any free radiolabelled nucleotides, but should contain purified RNA or RNA / radiolabelled cDNA, is measured.
  • the signal-to-noise ratio in the eluate from built-in labeled substances to non-built-in labeled substances in comparative reactions was calculated. If unlabeled nucleotides were added after the reaction but before the purification, the signal-to-noise ratio increased to 8 times.
  • 0.1 mM fluorophore-labeled nucleotides are incubated in water together with 0.4 ⁇ g DNA and 0.1 mM dNTP. Fluorophore-labeled nucleotides cannot be incorporated because no enzymes are added. These mixtures are briefly incubated and then supplemented with 10 ⁇ l of a mixture which contains unlabeled nucleotides (10 mM) in various reaction batches. 10 ⁇ l of water (0 mM dNTP) are added to each reaction mixture. This serves as a control approach.
  • All batches are cleaned using a silica cleaning step (e.g. "QiaQuick” from Qiagen, D-40724 Hilden).
  • a silica cleaning step e.g. "QiaQuick” from Qiagen, D-40724 Hilden.
  • the DNA binds to the silica membrane.
  • the optical density of the eluate is measured in the photometer under standard conditions.
  • nucleotides that are not labeled are added to the mixture after incubation, but before nucleic acid purification, the background contamination is reduced by up to 70 times.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Physical Or Chemical Processes And Apparatus (AREA)

Abstract

L'invention concerne un procédé pour la réduction de la contamination d'arrière-plan après une réaction de marquage.
EP03739498A 2002-02-15 2003-02-17 Procede pour la reduction de la contamination d'arriere-plan apres des reactions de marquage Ceased EP1476579A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10206616 2002-02-15
DE10206616A DE10206616A1 (de) 2002-02-15 2002-02-15 Verfahren zur Reduktion der Background-Kontamination nach Markierungsreaktionen
PCT/EP2003/001590 WO2003068981A2 (fr) 2002-02-15 2003-02-17 Procede pour la reduction de la contamination d'arriere-plan apres des reactions de marquage

Publications (1)

Publication Number Publication Date
EP1476579A2 true EP1476579A2 (fr) 2004-11-17

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP03739498A Ceased EP1476579A2 (fr) 2002-02-15 2003-02-17 Procede pour la reduction de la contamination d'arriere-plan apres des reactions de marquage

Country Status (4)

Country Link
US (1) US20050064481A1 (fr)
EP (1) EP1476579A2 (fr)
DE (1) DE10206616A1 (fr)
WO (1) WO2003068981A2 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2444695T3 (es) * 2006-06-23 2014-02-26 Alethia Biotherapeutics Inc. Polinucleótidos y polipéptidos implicados en el cáncer
CA2740900C (fr) 2008-11-03 2019-09-24 Alethia Biotherapeutics Inc. Anticorps qui bloquent specifiquement l'activite biologique d'un antigene tumoral
SI3173427T1 (sl) 2011-03-31 2019-08-30 ADC Therapeutics SA, Protitelesa proti antigenu 1, povezanemu z ledvicami, in antigen vezavni fragmenti le-tega
KR102102239B1 (ko) 2012-01-09 2020-04-21 에이디씨 테라퓨틱스 에스에이 유방암을 치료하기 위한 방법
US10023902B2 (en) * 2012-09-13 2018-07-17 Purdue Research Foundation Methods for detecting enzyme activity using fluorescence lifetime imaging

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6072043A (en) * 1996-06-04 2000-06-06 Polyprobe, Inc. Optimally fluorescent oligonucleotides
US6262252B1 (en) * 1997-05-19 2001-07-17 Mirus, Inc. Single-step method for labeling nucleic acids with mustard or aziridine labeling reagents
US6255476B1 (en) * 1999-02-22 2001-07-03 Pe Corporation (Ny) Methods and compositions for synthesis of labelled oligonucleotides and analogs on solid-supports

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO03068981A2 *

Also Published As

Publication number Publication date
WO2003068981A3 (fr) 2003-12-31
DE10206616A1 (de) 2003-09-04
WO2003068981A2 (fr) 2003-08-21
US20050064481A1 (en) 2005-03-24

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