EP1476579A2

EP1476579A2 - Method for reducing background contamination after marking reactions

Info

Publication number: EP1476579A2
Application number: EP03739498A
Authority: EP
Inventors: Christian Korfhage
Original assignee: Qiagen GmbH
Current assignee: Qiagen GmbH
Priority date: 2002-02-15
Filing date: 2003-02-17
Publication date: 2004-11-17
Also published as: US20050064481A1; WO2003068981A2; WO2003068981A3; DE10206616A1

Abstract

The invention relates to a method for reducing background contamination after a marking reaction.

Description

Process for reducing background contamination after

labeling reactions

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.

Labeling reactions for the quantification or identification of substances have become established in the prior art. For example

Nucleic acids are labeled with modified nucleotides. In other cases, labeled substances are used as a code or sensor to identify other molecules or to track processes.

In order to obtain pure labeled substances, appropriately labeled molecules are used as substrates in these labeling reactions. The incorporation of non-radioactive modification groups takes place through enzymatic, photochemical or chemical reactions. In contrast, radioactive isotopes are largely incorporated by enzymatic reactions. The marking positions and type of marking are different in the different methods, depending on whether radioactive isotopes or non-radioactive reporter groups are used.

In this way, nucleic acids (DNA, RNA) of various lengths can be labeled via the enzymatic incorporation of labeled nucleotides. As a result, probes can be produced which have a high marking density and a high sensitivity.

In the enzymatic labeling reactions either 5'-labeled primers (PCR labeling) or labeled nucleotides are used instead of unlabeled nucleotides or a combination of both methods.

The disadvantage inherent in all of these marking techniques is that the marked product has an annoying background contamination. This problem is the starting point of the present invention. A large number of cleaning protocols for removing undesired accompanying substances of the desired marked product are known from the prior art. However, these often have the disadvantage that contamination is ultimately still detectable when using the labeled substances.

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.

In numerous molecular biological applications, nucleic acid fragments, oligonucleotides, proteins and peptides are labeled, for example, with radioactive compounds or with dyes. However, 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 radioactive labeling is done by incorporating unstable isotopes or

Substances that contain unstable isotopes or by replacing stable natural isotopes with unstable isotopes [F. Lottsspeich and H. Zorbas (ed.), Bioanalytik, Spektrum Akademischer Verlag, Heidelberg, 1998]. The chemical structure is not changed by this isotope exchange, so that the labeled molecules have the same physicochemical properties as the natural substances. This means that the reaction conditions for the enzymatic incorporation of labeled nucleotides into hybridization probes need not be changed. In the case of the most commonly used ³² P or ³³ P phosphate label, the exchange position is either the a or γ position of the phosphate residues in 2'-deoxyribo, 3'-deoxyribo or 2'-ribonucleotides.

In the case of ³⁵ S, the exchange is made for an oxygen atom of the σ-phosphate.

The ²⁵ l mark is made at the C-5 position of the cytosine. In contrast to isotope labeling, when using non-radioactive molecules to modify probes, 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. To protect against non-specific interactions, 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 (DNA as well as RNA) are used in molecular biological cloning as a reagent or as a sample. In general, the labeling reagent can be covalently bound to the substance to be labeled or non-covalently associated with it.

In this case, 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. On the other hand, 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.

In the cases described, 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.

First, some of the labels of nucleic acids known from the prior art will be explained by way of example. In the sense of the present invention, modifications or variations of these methods as well as methodically other methods can also be used. 5'-phosphorylation of DNA

[Γ- ³² P] ATP is used for radioactive labeling of single-stranded hybridization samples or DNA for sequencing according to Maxam-Gilbert. Here, the γ-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.

Terminal transferase reaction

DNA oligonucleotides can be labeled enzymatically via the terminal transferase by attaching labeled dNTPs independently of the template (tailing).

If mixtures of labeled and unlabeled dNTPs are used, single-stranded chains (tails) are formed in a template-independent reaction and carry several labels. When using labeled cordyceptin triphosphate (3'-dATP) or 2 ', 3'-ddNTPs, only a single labeled nucleotide is attached since the reduced 3' position cannot be extended any further.

3 'end label of DNA

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. For this purpose, the [α-32P] deoxynucleoside triphosphates are used, which are complementary to the respective first base of the 5 'single-strand ends.

Radioactive labeling of DNA by nick translation In addition to small amounts of pancreatic DNase I, 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. This causes the nick to move in the δ '→ 3' direction (nick translation). Accordingly, this is a DNA synthesis (replacement synthesis), whereas in contrast to the random priming synthesis (see below), the yields remain less than 100% for the reasons described above, due to the reaction.

Labeling of DNA fragments by "random priminq"

In the "random priming" protocol, double-stranded DNA is first denatured. The

Rehybridization of both strands is prevented by cooling to low temperatures and by adding high concentrations of the primers. 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. Since statistically several primers bind per target, only partial sequences are replicated per primer elongation; a mixture of different probe lengths is created in this way, but the partial sequences are all target-specific and carry homogeneous labeling. Digoxigenin-labeled probes, which are generated by the "random priming" method, can be used to achieve high detection sensitivities in the sub-picogram range

Labeling of RNA by in wYro transcription

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 [α- ³² P] nucleotides (ATP or CTP).

Non-radioactive labeling of nucleic acids

When introducing non-radioactively labeled substances, chemical groups or components that are not part of nucleic acids conjugate with the sample via enzymatic or other chemical reactions. After hybridization with the nucleic acid, suitable indicator systems or detect

Detection systems the modified groups of the sample.

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.

After hybridization, the 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.

Enzymatic methods

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.

Photochemical labels

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

chemiluminescence

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.

fluorescent labeling

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. at

Fluorophore labeling is widely used in nucleic acids in hybridization systems such as in microarrays.

As already mentioned at the beginning, 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.

In addition to the radioactive markers or reporter groups already mentioned - among which ³ H, ¹⁴ C, ³² P, ³³ P, ³⁵ S or ¹²⁵ l are preferred - non-radioactive markers can also be used according to the invention. Such 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.

So-called. Metal markers, e.g. metal-marked - especially Au- and Ag-marked

Antibody.

Enzyme markers for direct enzyme coupling, e.g. Alkaline phosphatase (AP),

Horseradish peroxidase (POD), microperoxidase,? -Galactosidase, urease, glucose-

Oxidase, glucose-6-phosphate dehydrogenase, hexokinase, bacterial luciferase,

Firefly luciferase.

Enzyme markers for enzyme substrate transfer, e.g. Glucose oxidase: horseradish

Peroxidase.

Enzyme markers for enzyme complementation, e.g. Inactive? -Galactosidase: σ- peptide.

Polymeric markers, e.g. Latex dye particles or polyethyleneimine.

In general, 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. In all of the above-mentioned methods, the task is to avoid a high background signal, which leads to a short range of sensitivity and dynamic range of the measurements.

According to the invention, 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.

This addition is followed by a cleaning process known per se from the prior art. This can be any method that leads to a purification of the labeled substance.

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.

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.

4 shows a 70-fold reduction in the background for example 3.

Further details of the invention are explained in the examples.

Examples

Example 1: Radioactive labeling

In this experiment, the background contamination of the radioactive nucleotides is measured after purification.

10 μCi of ³² P-dCTP together with 1 μg poly (A) RNA, standard buffer, which buffers in a pH range from 7 to 10 (for example RT buffer from Qiagen, D-40724 Hilden), 0 , 1 mM (mmol / L) dNTP, 10 U RNase inhibitor (Promega) and 1μM oligo-dT15.

No radiolabelled nucleotides can be incorporated during this incubation because no enzymes are added. The mixture is then incubated at 37 ° C. for 1 h. After incubation, 10 μl of a mixture which contains unlabelled nucleotides of different concentrations are added to the mixture in different reaction batches. A reaction mixture to which 10 μl of water (0 mM dNTP) was added serves as a control mixture.

These 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. Here, 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.

Example 2: Radioactive labeling

In this experiment, the ratio of signal to noise of built-in labeled substances to non-built-in labeled substances is measured in comparison reactions.

10 μCi of ³² P-dCTP together with 1 μg poly (A) RNA, standard buffer, which buffers in a pH range from 7 to 10 (for example RT buffer from Qiagen, D-40724 Hilden), 0 , 1 mM dNTP, 10 U RNase inhibitor (Promega) and 1μM oligo-dT15.

Some of the 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 μl of a mixture which contains unlabelled nucleotides of different concentrations in different reaction batches. 10 μl 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). In the control reaction, 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.

Example 3: Fluorescent labeling

In this experiment, the background contamination of nucleotides that are labeled with fluorophore is measured after purification.

0.1 mM fluorophore-labeled nucleotides are incubated in water together with 0.4 μg 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 μl of a mixture which contains unlabeled nucleotides (10 mM) in various reaction batches. 10 μl 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).

During the cleaning process, the DNA binds to the silica membrane. The optical density of the eluate is measured in the photometer under standard conditions.

If 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.

Claims

claims

1. A method for labeling biomolecules, preferably peptides, proteins and nucleic acids, wherein the biomolecules are reacted with a labeling reagent and unreacted labeling reagent is separated from the labeled substance, characterized in that prior to the purification of the labeled biomolecules, preferably before Adds an unlabeled substance, preferably an unlabelled derivative and particularly preferably the corresponding unlabelled starting material, to the reaction mixture at the end of the labeling reaction, particularly preferably in the last third and very particularly preferably at the end of the labeling reaction.

2. The method according to claim 2, characterized in that the nucleic acids comprise DNA (DNA) and / or RNA (RNS).

3. The method according to claim 1 or 2, characterized in that the labeling agent is a radioactive isotope, preferably ³ H, ¹⁴ C, ³² P, ³³ P, ³⁵ S or ¹²⁵ l.

4. The method according to claim 1 or 2, characterized in that the marking agent is a fluorescent marker.

5. The method according to claim 4, characterized in that the fluorescent marker fluorescein (FITC, FLUOS), rhodamine (RHODOS, RESOS,

RESIAC), Hydroxycoumarin (AMCA), Benzofuran, Texas Red, Biman, and / or Ethidium / Tb ³⁺ .

6. The method according to claim 4, characterized in that the fluorescent marker is a fluorescent marker for time-triggered fluorescence, preferably a lanthanoid (Eu ³ 7 Tb ³⁺ ) complex, a micelle or a chelate.

7. The method according to claim 4, characterized in that the fluorescent marker is a fluorescent marker for fluorescence energy transfer, preferably fluorescein: rhodamine.

8. The method according to claim 1 or 2, characterized in that the marking agent is a luminescent marker.

9. The method according to claim 8, characterized in that the luminescence marker is a luminescence marker for chemiluminescence, preferably a (/ so) luminol derivative or acridine ester.

10.The method according to claim 8, characterized in that the

Luminescence marker is a luminescence marker for electroluminescence, preferably a Ru ²⁺ - (2,2'-bipyridyl) ₃ complex.

11. The method according to claim 8, characterized in that the

Luminescence marker is a luminescence marker for the luminescence energy transfer, preferably rhodamine: luminol.

12. The method according to claim 1 or 2, characterized in that the labeling agent is a metal marker, preferably a metal-labeled and particularly preferably an Au-labeled or Ag-labeled antibody.

13. The method according to claim 1 or 2, characterized in that the labeling agent is an enzyme marker.

14. The method according to claim 13, characterized in that the enzyme marker is an enzyme marker for direct enzyme coupling, preferably alkaline phosphatase (AP), horseradish peroxidase (POD), microperoxidase, β-galactosidase, urease, glucose oxidase, glucose-6 Phosphate dehydrogenase, hexokinase, bacterial luciferase, firefly luciferase.

15. The method according to claim 13, characterized in that the enzyme marker is an enzyme marker for the enzyme substrate transfer, preferably glucose oxidase: horseradish peroxidase. 16. The method according to claim 13, characterized in that the enzyme marker is an enzyme marker for enzyme complementation, preferably an inactive β-galactosidase: peptide.

17. The method according to claim 1 and 2, characterized in that

Marking agent is a polymeric marker, preferably latex dye particles or polyethyleneimine.

18Nerfahren according to any one of claims 1 to 2, characterized in that the labeling agent covalently to the peptide to be labeled, protein or

Nucleic acid is bound or is non-covalently associated.

19. The method according to claim 2, characterized in that the conversion of DNA polymerase, RNA polymerase or reverse transcriptase is catalyzed.

20.The method according to any one of claims 1 to 19, characterized in that the concentration ratio of the unlabelled reagent to the labeled reagent is in an interval of 1: 1 to 1000: 1, preferably in an interval of 10: 1 to 100: 1 ,