EP0983379A1

EP0983379A1 - Method and kit for the detection of mutations in dna's using restriction enzymes

Info

Publication number: EP0983379A1
Application number: EP97919050A
Authority: EP
Inventors: Ursula Bilitewski; Dirk Kuhlmeier
Original assignee: Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Current assignee: Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Priority date: 1997-05-21
Filing date: 1997-09-10
Publication date: 2000-03-08
Also published as: AU737771B2; AU4302297A; WO1998053098A1; CA2290510A1; RU2193069C2; JP2001525678A

Abstract

The invention relates to a method for detecting DNA mutations using restriction enzymes. One condition to implement said method is that the cleaving performance of the selected restriction enzyme changes due to mutation. The relevant DNA segment in the genome containing the mutation is multiplied by means of polymerase chain reaction (PCR), wherein the primers used can each be labeled. The first primer enables the segment to be bonded to the surface of the carrier; the label of the second primer is used to enable detection. A restriction enzyme is incubated with the PCR amplicon, wherein the mutated segment is not cleaved if the cleavage site is suppressed by mutation. After the segments are bonded to the surface of the carrier, a detection reaction takes place, wherein the marking label of the second primer is removed in the wild type, insofar as it can be identified in the mutant. A plurality of labels enabling direct or indirect detection is possible. This provides a rapid method for detecting specific mutations in the genome without resorting to complex conventional methods.

Description

Method and kit for the detection of mutations in DNA 's with the help of restriction enzymes

The present invention relates to a method for the detection of mutations of DNAs, a DNA being amplified by means of a PCR and the amplicons being cut and detected with one or more restriction enzymes, characterized in that two labeled primers are used, the labeling of the first Primer for binding the amplicons to one or more carriers and the marking of the second primer is used for detection. The present invention further relates to a kit for carrying out the method described above, which is characterized in that it comprises two labeled primers. In the course of the elucidation of the human genome, the importance of gene defects in the development of a large number of diseases becomes apparent. For example, there are changes in genetic information in many types of cancer, cystic fibrosis, Alzheimer's disease and in the development of thrombosis, which may make disease possible. The detection of these mutations is therefore becoming increasingly important in medical diagnostics.

Mutations are currently often detected using restriction analysis. Restriction enzymes are used here, which recognize double-stranded DNA in a sequence-specific manner and can cut at the appropriate place. Approximately 500 different restriction enzymes with over 100 different interfaces are currently characterized. This high specificity is used to detect the defects. Because a mutation that triggers a pathological change often lies in the region of such an enzyme interface. The mutation changes the interface so that the strand to be examined is not cut. In other cases, a mutation first creates a possible interface for an enzyme that was not previously available.

For standard analysis, the genomic DNA to be examined is subjected to a polymerase chain reaction (PCR), with the aid of which a desired piece of DNA on which the mutation lies can be reproduced. For this, so-called primers, sequence pieces, are used, which define the beginning and the end of the amplified piece of DNA. You also need free nucleotides, building blocks of DNA, and polymerase, an enzyme that prevents the formation of the catalyzing searching and reproducing DNA segments. To the

When the PCR is running, a thermal cycler is also required, a device that realizes the required temperature profile for a PCR reaction.

Following the duplication of the segment to be examined, the above-mentioned degradation takes place with the aid of the restriction enzyme. Depending on the mutation, this cuts the DNA in a sequence-specific manner under defined conditions or there is no cut. Depending on the concentration of the enzyme, this process can take a few hours.

Up to now, agarose gel electrophoresis has been used to analyze the product: an agarose polymer is dissolved in a buffer solution by heating and after a cooling phase, the gelling polysaccharide is poured into a device in which the electrophoresis is to be carried out. When the gel has cooled completely, the samples are applied and the size-dependent migration of the DNA fragments in the agarose gel occurs with the application of a potential difference.

The fragments can be visualized e.g. with the addition of fluorescent intercalates, substances that are embedded in the DNA and are visible under UV light. A mutation can be diagnosed if a specific change in the restriction pattern becomes apparent in the gel.

In addition to the duplication of the relevant DNA section, the elaborate implementation of electrophoretic methods, linked to the preparation, is therefore used to detect a mutation of gels, application of samples and corresponding waiting time at the

Course of gel electrophoresis.

These processes are very time-consuming, cumbersome and therefore expensive.

The invention is therefore based on the object of developing a method and a kit with which the mutation forms described above can be detected simply, reproducibly and with a high sample throughput, the disadvantages of gel electrophoresis being avoided.

This object is achieved by a method for the detection of mutations of DNAs, wherein a DNA is amplified by means of a PCR and the amplicons are cut and detected (analyzed) with one or more restriction enzymes, which method is characterized in that two marked primers are used and the marking of the first primer is used to bind the amplicons (duplicated DNA segments) to one or more supports and the marking of the second primer is for detection (analysis).

The PCR used in this method can be carried out as usual, the primers described above being used as primers.

According to the invention, preference is given to using supports which, even under the conditions of a PCR, enable the first primers to be bound. Carriers that have an activated surface, such as, for example, have proven to be particularly advantageous the appropriately treated surface of the reaction vessel or a microtiter plate.

In the method according to the invention, the first primers are partially or completely fixed to the supports before, during or after the PCR, while the second primers are preferably bound free or not to the support and are optionally present in excess in the reaction solution. The primers can be added to the reaction solution simultaneously or in succession.

The second primers should preferably have labels that can be easily detected by conventional methods when they are detected.

According to the invention, one or more restriction enzymes can be added before, during or after the binding of the DNA segments (amplicons from the PCR) to the carrier, the addition after the binding being preferred. According to the invention, the restriction enzymes can be incubated with the amplicons.

All known restriction enzymes can be used as restriction enzymes, it of course depending on the sequence to be cut which enzyme (s) are used in the specific case.

After the amplicons have been bound to suitable supports by means of the first primers and before and / or after cutting, preferably after cutting the amplicons with one or more of the restriction enzymes described above, the amplicons can be washed with a suitable dishwashing detergent, such as, for example, tem or distilled water, the optional additives such as

Contains buffer to be rinsed. When rinsing is performed after cutting, cut, unfixed segments are removed.

According to the invention, the detection methods are selected so that the respectively selected marking (the label) of the second primer can be detected. The detection can be carried out directly or indirectly according to the marking of the second primer.

There are a variety of possible methods of biochemical analysis that are known from practical use:

1. Coupling with spectroscopically directly detectable substances.

2. Labeling with enzyme labels that convert their substrate into photochemically detectable substances. The associated amplification of the measurement signal is advantageous.

According to a first embodiment (see scheme 1), the DNA is first amplified and the amplicon is then bound to a suitable support, the label on the first primer serving as the coupling mediator. If necessary, the first primers can also have several corresponding labels.

According to a second embodiment (see Scheme 2) of the present invention, the first primers are first bound to a suitable support such as the support surface of the vessel in which the PCR reaction takes place. A coupling method must be selected that meets the constraints of the PCR, such as temperature stability. The reaction then proceeds according to Standard conditions, wherein the second primer is not bound to the support and is preferably present in excess in relation to the first primer.

During the reaction, the desired DNA segment is formed in the solution, but hybridization and amplification also take place on the surface of the support, initiated by the bound primer. At the end of the PCR, the desired products are obtained which are bound directly to the support. Excess PCR starting materials (e.g. free nucleotides etc.) and unbound products are removed in a rinsing step. The segment is then cleaved by the enzyme, optionally a further rinsing step and the detection as described above.

For example, in Scheme 1, you can labein with biotin. In step 3 of this scheme, the coupling to the support, for example a microtiter plate, could then be carried out via a biotin-binding protein, for example via streptavidin or avidin, which would be adsorbed or covalently bound, for example on the surface of the wells the microtiter plate. Microtiter plates coated with streptavidin are commercially available (Boehringer Mannheim).

In scheme 2, a covalent coupling to the support could be provided for primer 1, for example on a microtiter plate. For this purpose, phosphorylated primers are suitable, for example, which could react in a carbodiimide reaction with appropriately activated carriers. An example of suitable functional groups of the support, for example the microtiter plates, would be amino groups. Such microtiter plates are also commercially available.

The same labels for primer 2 can be used in scheme 1 and in scheme 2. Examples of these are fluorescein, fluorescein derivatives, rhodamine, rhodamine derivatives, Cy5 (fluorescent dye), Cy3 (fluorescent dye) or compounds which can be detected indirectly.

Fluorescent dyes: These primers can then be directly detected using a suitable fluorimeter.

Digoxin: indirect detection can be performed by adding an (enzyme-labeled) antibody. Biotin: Indirect detection can be performed by adding an avidin / enzyme conjugate, for example by adding avidin or streptavidin.

The binding of the respective recognition protein (antibody or (strept) avidin) can either be detected, as is customary in other microtiter plate assays, by adding suitable enzyme substrates, with peroxidase or alkaline phosphatase being suitable enzymes (enzyme reaction provides colored products), or else it can then be followed directly if the coupling is not made on microtiter plates, but on suitable sensors. Suitable sensors can be, for example, planar waveguides, as are known from biosensor technology, and on which the same coupling reactions can be carried out with the aid of primer 1 as have been described above for microtitre plates. According to the invention, the DNA to be examined can have an interface as a wild type or as a mutant.

a) The interface is intact DNA (wild type):

If a mutation occurs in the relevant area of the DNA, the interface is removed in the selected example. A point mutation is sufficient to prevent the enzyme from specifically recognizing and cutting the segment. The restriction enzyme, PCR starting materials and, if appropriate, separated PCR products which contain the detectable label can be removed by a rinsing step before the actual detection.

The detection step analyzes whether the label is present or not. If there is a mutation, the detectable label is present and a reaction is obtained depending on the selected label. In the case of enzyme labeling one would e.g. observe a catalyzed color reaction. If there is no mutation, the enzyme can cut specifically and the label is removed. There is no detectable signal.

b) The interface exists with mutated DNA:

If a mutation occurs in the relevant area of the DNA, the interface is created in the selected example. A point mutation can be sufficient for the enzyme to specifically recognize and cut the segment. By a rinsing step can prior to the actual detection restriction enzyme, PCR reactants and any separated PCR products that contain the detectable La ^¬ bel be removed. The detection step analyzes whether the label is present or not. If there is a mutation, the detectable label is not present and no reaction is obtained. If there is no mutation, the enzyme cannot cut and the label is not removed. A detectable signal is obtained.

The invention is explained below with reference to the illustrations for case a):

Scheme 1 illustrates the sequence of the first assay setup according to the invention. Fig. 1 shows the amplification of the target using the labeled primers. The PCR is carried out in suitable containers. Figure 2 shows the PCR reaction products labeled with labein. One end of the reaction products has a label that is used for later binding to a support surface and the label at the other end is used for detection. The searched sequence, which can contain a potential mutation, is within the amplified segments.

3 shows the binding of the segments made possible by the first marking; in Fig. 4 it is examined by the action of a restriction enzyme whether the interface of this enzyme has been removed by a mutation or not. If a mutation is present, as shown in FIG. 5, the interface is omitted and the detectable labels are not removed. If there is no mutation, the enzyme can cut. The separated segment is removed by a simple washing step and consequently no signal is obtained at the bound part of the segment. Scheme 2 demonstrates the second structure of the assay according to the invention. 1 shows that part of the first primer, which is used for binding to the support surface, is already bound before the start of the PCR. The second primer, which can be detected, is in excess in the solution. 2 indicates that after the PCR reaction has ended, the amplicon, which contains the sequence to be examined, is surface-fixed. Subsequently, the same restriction enzyme is added (FIG. 3), which, as described above, cuts the wild type, but not the mutation sequence. The result of the assay carried out is illustrated in FIG. 4. After a washing step (not shown), the detectable label is still present on the fixed segment in the event of a mutation. In the wild type, however, no signal is obtained since the enzyme cuts due to the existing interface and, as a result, the separated, labeled segment can be removed by a washing step.

According to the invention, a kit for carrying out the methods disclosed above is provided, which is characterized in that it comprises two labeled primers, it preferably additionally containing free nucleotides and a polymerase.

The first primer is suitable for binding PCR reaction products to one or more supports and the second primer is used for detection, the support preferably comprising an activated surface.

The method according to the invention and the kit according to the invention have the following advantages over the prior art: - The described embodiments enable time savings compared to gel electrophoretic detection. The second embodiment in particular has advantages since the PCR and the analysis of the products take place in one container. Repipetting is no longer necessary; the handling of the samples is simplified and the associated sources of error are eliminated.

- Depending on the choice of the label to be detected, the mutation is detected immediately after cleavage by the enzyme and not only after gel electrophoresis.

- In the clinical application of test procedures one often has to deal with a high sample volume. The effort to carry out gel electrophoresis to deal with the amount of sample is correspondingly large. Since the new method can be carried out entirely in microtiter plate format, e.g. 96 or 384 samples can be measured in parallel.

- Gel electrophoresis uses toxic DNA intercalation compounds, which can be completely dispensed with in the new method.

example 1

The mutation in protein factor V is held responsible for an increased risk of thrombosis, since it inhibits the breakdown of glycoprotein factor V by the serine protease APC (activated protein C). Human factor V is a 330 kDa glycoprotein that interacts with APC and normally cleaved by APC at a very specific point in the sequence: in the human protein behind Arg 506 (arginine as the 506th amino acid in the protein). In sequence studies in the associated gene, it was found that the exchange of 1.691 G (G = guanine) for A (adenine) in the nucleotide sequence correlates with an increased risk of thrombosis. This mutation means that the Arg 506 (product of the nucleotide sequence CGA) in the protein is replaced by glutamine (gin as the product of the nucleotide sequence CAA) and the cleavage by the protease described above therefore no longer takes place. To detect this mutation, primers are used which are used to amplify a region of the gene which comprises the corresponding nucleotide sequence by means of a PCR reaction. For example, fragments of 267 base pairs in length were obtained. Treatment with the restriction enzyme Mnl 1 gives rise to 3 fragments with 67, 37 and 163 base pairs (bp), if 1,691 guanine is present at the site, since the sequence specificity enables the enzyme to cleave the gene fragment at 2 sites (after 67 bp and after another 37 bp). If the mutation to adenine is present, the second cleavage site is omitted, so that only two cleavage products are observed (67 bp and 200 bp length), since the sequence has changed as a result of the mutation so that the enzyme used can no longer work. The various fragments are detected using gel electrophoresis. This information was mainly obtained from the publication by RM Bertina et al. , Mutation in blood coagulation factor V associated with resistance to activated protein C, Nature, 369, 1994, 64-67.

Claims

claims

1. A method for the detection of mutations of DNA_s, wherein a DNA is amplified by means of a PCR and the amplicons are cut and detected with one or more restriction enzymes, characterized in that two labeled primers are used, the labeling of the first primers for binding the amplicons to one or more supports and the marking of the second primer is used for detection.

2. The method according to claim 1, characterized in that carriers are used to which the first primers can also be bound under the conditions of a PCR.

3. The method according to any one of the preceding claims, characterized in that the carriers have an activated surface.

4. The method according to any one of the preceding claims, characterized in that the first primers are partially or completely fixed to the supports before, during or after the PCR and the second primers are optionally present freely in the reaction solution.

5. The method according to any one of the preceding claims, characterized in that one or more restriction enzymes are added before, during or after the binding of the amplicons to the carrier.

6. The method according to any one of the preceding claims, characterized in that the restriction enzymes are incubated with the amplicons.

7. The method according to any one of the preceding claims, characterized in that after cutting with the restriction enzymes is optionally rinsed and the markings of the second primer are detected.

8. The method according to any one of the preceding claims, characterized in that the detection method is selected according to the marking of the second primer.

9. The method according to any one of the preceding claims, characterized in that first an amplification and then a binding of the amplicons to a suitable carrier is carried out.

10. The method according to any one of claims 1 to 8, characterized in that first the first primers are bound to a suitable support and then an amplification of the DNA is carried out with the second primer.

11. The method according to any one of the preceding claims, characterized in that the detection can be carried out directly or indirectly according to the marking of the second primer.

12. Kit for performing a method according to any one of the preceding claims, characterized in that it comprises two labeled primers.

13. Kit according to claim 12, characterized in that it additionally comprises free nucleotides, a polymerase and at least one restriction enzyme.

14. Kit according to one of the preceding claims, characterized in that the first primers are suitable for binding PCR reaction products to one or more supports and the second primers are used for detection.

15. Kit according to any one of the preceding claims, characterized in that it comprises a suitable carrier, preferably with an activated surface.

16. Use of a kit according to one of claims 11 to 15 for the detection of mutations.