AU4302297A - Method and kit for the detection of mutations in DNA's using restriction enzymes - Google Patents

Description

Method and kit for the detection of mutations in DNAs using restriction enzymes The present invention relates to a method for the detection of mutations of DNAs, wherein a DNA is amplified by means of PCR and the amplicon is cleaved and detected with one or more restriction enzymes, characterised in that two labelled primers are used, the label of the first primers serving to bind the amplicons to one or more supports and the label of the second primers serving for detection. The present invention further relates to a kit for carrying out the method described above, which kit is characterised in that it comprises two labelled primers. In the course of mapping the human genome, the significance of genetic defects in the development of a large number of disor ders has become apparent. For example, in the case of many types of cancer, cystic fibrosis, Alzheimer's disease and in the development of thromboses, changes are present in the genetic information which may make it first possible for a disease to develop. The detection of those mutations is there fore gaining increasing importance in medical diagnostics. At present, mutations are frequently detected by means of restriction analysis. This makes use of restriction enzymes, which are able to recognise double-stranded DNA sequence specifically and cleave it at the appropriate site. So far, approximately 500 different restriction enzymes with more than 100 different cleavage sites have been characterised. That high specificity is used for the detection of defects, since often a mutation that triggers a pathological change lies in the region of such a cleavage site of an enzyme. As a result of the mutation, the cleavage site is altered in such a way that the strand to be investigated is not cleaved. In other ases, it is only as a result of a mutation that a possible -2 cleavage site for an enzyme is produced, which site was not present before. For standard analysis, the genomic DNA to be investigated is subjected to a polymerase chain reaction (PCR) by means of which a desired fragment of DNA on which the mutation lies can be amplified. So-called primers, sequence fragments that define the start and end of the amplified fragment of DNA, are used for this. Free nucleotides, building blocks of DNA, and polymerase, an enzyme that catalyses the formation of the DNA segments to be investigated and amplified, are also required. For the PCR to proceed a thermocycler is also required, that is to say, an apparatus that produces the required temperature profile for a PCR reaction. Following amplification of the segment to be investigated, the above-mentioned fragmentation by means of the restriction enzyme takes place. Depending upon mutation, the latter cleaves the DNA sequence-specifically under defined conditions, or no cleavage occurs. That process may take a number of hours, depending upon the concentration of the enzyme. For analysis of the product, agarose gel-electrophoresis has previously been used: for this, an agarose polymer is dissolved in a buffer solution by heating and, after a cooling phase, the gelling polysaccharide is poured into an apparatus in which the electrophoresis is to be carried out. Once the gel has cooled completely, the samples are applied and, with the application of a potential difference, size-dependent migration of the DNA fragments in the agarose gel takes place. Visualisation of the fragments is achieved, for example, with the addition of fluorescent intercalates, substances that intercalate themselves in the DNA and are visible under -3 UV-light. A mutation can be diagnosed when a specific change in the restriction pattern is discernible in the gel. For the detection of a mutation, therefore, in addition to amplification of the relevant DNA segment, expensive electro phoretic methods are carried out, involving the preparation of gels, application of samples and a suitable waiting period while the gel-electrophoresis proceeds. Those methods are very time-consuming, complicated and there fore costly. The problem underlying the invention is therefore to develop a method and a kit with which the forms of mutation described above can be detected simply, reproducibly and with a high sample throughput, while avoiding the disadvantages of gel electrophoresis. That problem is solved by a method for the detection of muta tions of DNAs wherein a DNA is amplified by means of PCR and the amplicons are cleaved and detected (analysed) with one or more restriction enzymes, which method is characterised in that two labelled primers are used and the label of the first primers serves to bind the amplicons (amplified DNA segments) to one or more supports and the label of the second primers serves for detection (analysis). The PCR used in that method may be carried out as normal, using as primers the primers described above. There are preferably used according to the invention supports that allow binding of the first primers also under the condi tions of PCR. Supports that have an activated surface, such as, for example, the appropriately treated surface of the reac ill tion vessel or a microtitre plate, have been found to be espe cially advantageous. In the method according to the invention, the first primers are partially or completely fixed to the supports before, during or after the PCR, whereas the second primers are preferably free, that is to say not bound to the support, and are present in the reaction solution optionally in excess. The primers may be added to the reaction solution simultaneously or in succession. The second primers are preferably to have labels that can be detected easily by usual methods during a detection operation. According to the invention, one or more restriction enzymes may be added to the supports before, during or after binding of the DNA segments (amplicons from the PCR), addition after binding being preferred. According to the invention, the restriction enzymes may be incubated with the amplicons. All known restriction enzymes may be used as the restriction enzymes, the enzyme(s) used in a particular case naturally depending upon the sequence to be cleaved. After binding of the amplicons to suitable supports by means of the first primers and before and/or after cleavage, preferably before cleavage of the amplicons with one or more of the above mentioned restriction enzymes, the amplicons may be washed with a suitable washing agent, such as, for example, optionally de ionised or distilled water optionally containing additives such as buffers. In the case where washing is carried out after the cleavage operation, cleaved non-fixed segments are removed. According to the invention, the detection methods are so selec ted that the particular label chosen for the second primers can - 5 be detected. Detection according to the label of the second primers can be carried out directly or indirectly. There are a large number of possible methods of biochemical analysis that are known from practical usage: 1. Coupling with spectroscopically directly detectable sub stances. 2. Labelling with enzyme labels that convert their substrate into photochemically detectable substances. The associated intensification of the measuring signal is advantageous in this case. In accordance with a first embodiment (see Scheme 1), first amplification of the DNA is carried out and then the amplicons are bound to a suitable support, the label on the first primer serving as a coupling mediator. The first primers may option ally also have several appropriate labels. In accordance with a second embodiment (see Scheme 2) of the present invention, first the first primers are bound to a suit able support, such as the support surface of the vessel in which the PCR reaction takes place. In that case, a coupling method that complies with the constraints of PCR, for example temperature stability, is to be chosen. The reaction subsequently proceeds according to standard conditions, the second primer not being bound to the support and preferably being present in excess in relation to the first primer. During the reaction, the desired DNA segment is formed in solu tion, but hybridisation and amplification also takes place at 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. In a washing step, excess PCR starting materials (for example free nucleotides etc.) and

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-6 unbound products are removed. Cleavage of the segment by the enzyme, optionally a further washing step, and the detection described above then take place. For example, in the case of Scheme 1 labelling can be carried out with biotin. In step 3 of that Scheme, coupling to the support, for example a microtitre plate, could then be carried out by way of a biotin-binding protein, for example by way of streptavidin or avidin, which would be adsorbed or covalently bound at the surface of the support, for example at the surface of the wells of a microtitre plate. Microtitre plates coated with streptavidin are commercially available (Boehringer Mann heim). In the case of Scheme 2, covalent coupling to the support, for example to a microtitre plate, could be provided for Primer 1. For that purpose there are suitable, for example, phosphoryl ated primers that could react with correspondingly activated supports in a carbodiimide reaction. An example of suitable functional groups of the support, for example of the microtitre plates, would be amino groups. Such microtitre plates also are commercially available. The same labels for Primer 2 may be used in the case of Scheme 1 and in the case of Scheme 2. Examples of these are fluorescein, fluorescein derivatives, rhodamine, rhodamine derivatives, Cy5 (fluorescent dye), Cy3 (fluorescent dye) or compounds that are detectable indirectly. Fluorescent dyes: in the case of these primers, direct subse quent detection can be carried out with a suitable fluorimeter. Digoxin: indirect detection can be carried out by the addition of an (enzyme-labelled) antibody. Biotin: indirect detection can be carried out by the addition of an avidin/enzyme conjugate, for example by the addition of k -avidin or streptavidin.

-7 The binding of the particular recognition protein (antibody or (strept)avidin) can either be detected as is customary in other microtitre plate assays, by means of the addition of suitable enzyme substrates, peroxidase or alkaline phosphatase being suitable enzymes (enzyme reaction yields coloured products), or, if coupling to suitable measuring sensors rather than to microtitre plates has been carried out, it can be observed directly. There may be mentioned as suitable measuring sensors, for example, planar waveguides as are known from biosensory technology and to which it is possible to carry out the same coupling reactions by means of Primer 1 as those desc ribed above for microtitre plates. According to the invention, the DNA to be investigated may have a cleavage site as a wild type or as a mutant. a) The cleavage site is present in intact DNA (wild type): If a mutation occurs in the relevant region of the DNA, the cleavage site in the example chosen is removed. Point mutation is sufficient to prevent the enzyme from specifically recognis ing and cleaving the segment. By means of a washing step, the restriction enzyme, PCR starting materials and, as the case may be, PCR products containing the detectable label which have been separated can be removed before the actual detection operation. In the detection step, analysis is carried out as to whether the label is present or not. If a mutation is present, the detectable label is present and a reaction is obtained accord ing to the label chosen. In the dase of an enzyme label, for example, a catalysed colour reaction would be observed. If no mutation is present, the enzyme is able to cleave specifically nd the label is removed. No detectable signal is obtained.

- 8 b) The cleavage site is present in mutated DNA: If a mutation occurs in the relevant region of the DNA, the cleavage site is produced in the example chosen. Point muta tion may be sufficient for the enzyme to recognise and cut the segment specifically. By means of a washing step, the restric tion enzyme, PCR starting materials and, as the case may be, PCR products containing the detectable label which have been separated can be removed before the actual detection operation. In the detection step, analysis is carried out as to whether the label is present or not. If a mutation is present, the detectable label is not present and no reaction is obtained. If no mutation is present, the enzyme is unable to cleave and the label is also not removed. A detectable signal is ob tained. The invention is explained in detail below with reference to the diagrams for Case a): Scheme 1 illustrates how the first assay arrangement according to the invention proceeds. Fig. 1 shows the amplification of the target by means of the labelled primers. PCR is carried out in vessels suitable for the purpose. Fig. 2 shows the PCR reaction products labelled with labels. One end of the reac tion products has a label that serves for subsequent binding to the surface of a support, and the label at the other end serves for detection. The sequence sought, which may contain a poten tial mutation, is located within the amplified segments. Fig. 3 shows the binding of the segments, made possible by the first label; in Fig. 4, the action of a restriction enzyme is used to investigate whether or not the cleavage site of that enzyme has been removed as a result of a mutation. If a muta lit! -9 tion is present, as shown in Fig. 5, the cleavage site is not present and the detectable labels are not removed. If no muta tion is present, the enzyme is able to cleave. By a simple washing step the separated segment is removed and consequently no signal is obtained at the bound part of the segment. Scheme 2 demonstrates the second arrangement of the assay in accordance with the invention. Fig. 1 shows that some of the first primer, which serves for binding to the surface of a support, is present in bound form even before the beginning of the PCR. The second primer, which is detectable, is present in excess in the solution. Fig. 2 indicates that, after the PCR reaction has been completed, the amplicon, which'contains the sequence to be investigated, is fixed to the surface. Restric tion enzyme is then added thereto (Fig. 3), which, as described above, cleaves the wild type but not the mutated sequence. Fig. 4 illustrates the result of the assay carried out. After a washing step (not shown), the detectable label is still pres ent on the fixed segment in the case of a mutation. In the case of the wild type, however, no signal is obtained since, owing to the cleavage site which is present, the enzyme is able to cleave and, as a result, the separated, labelled segment can be removed by a washing step. According to the invention a kit for carrying out the method disclosed above is provided, which kit is characterised in that it comprises two labelled primers and preferably contains, in addition, free nucleotides and a polymerase. The first primer is suitable for binding PCR reaction products to one or more supports and the second primer serves for detec tion, the support preferably having an activated surface. The method according to the invention and the kit according to invention have the following advantages over the prior art:

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- 10 - the embodiments described make it possible to save time in comparison with detection by gel-electrophoresis. The second embodiment in particular has advantages, since the PCR and the analysis of the products take place in one vessel. As a result, transfer by pipette is no longer necessary; the hand ling of the samples is simplified and sources of error associ ated therewith are excluded. - depending upon the choice of the label to be detected, detec tion of the mutation takes place immediately after cleavage by the enzyme rather than only after gel-electrophoresis has been carried out. - in the clinical application of test methods, a high sample load is often being dealt with. The expenditure on carrying out gel-electrophoreses to cope with the amount of samples is correspondingly high. Since the new method can be carried out entirely in a microtitre plate format, it is advantageously possible to measure, for example, 96 or 384 samples in parallel. - in the case of gel-electrophoresis, toxic DNA intercalating compounds are used, which can be completely dispensed with in the case of the new method. Example 1 Mutation in protein factor V is held responsible for an in creased risk of thrombosis since it inhibits degradation of the glycoprotein factor V by serine protease APC (activated protein C). Human factor V is a glycoprotein of 330 kDa that undergoes specific interactions with APC and is normally cleaved by APC at a very specific site in the sequence: in the case of the an protein, after Arg 506 (arginine being the 506th amino

(C

- 11 acid in the protein) . During sequence investigations in the associated gene, it was found that, in the nucleotide sequence, the exchange of 1,691 G (G = guanine) for A (adenine) corre lates with an increased risk of thrombosis. That mutation has the result that, in the protein, the Arg 506 (product of the nucleotide sequence CGA) is replaced by glutamine (Gln as the product of the nucleotide sequence CAA) and therefore the above-described cleavage by the protease no longer takes place. To detect that mutation primers are used with which a region of the gene comprising the corresponding nucleotide sequence is amplified by means of a PCR reaction. In that reaction, for example, fragments 267 base pairs long are obtained. By treat ment with the restriction enzyme MnlI 3 fragments of 67, 37 and 163 base pairs (bp) are produced therefrom when 1,691 guanine is present at the site since, owing to its sequence specific ity, the enzyme is able to effect cleavage of the gene fragment at two sites (after 67 bp and after a further 37 bp). When the mutation to adenine is present, the second cleavage site is not present, so that only two cleavage products are observed (67 bp and 200 bp in length) since, as a result of the mutation, the sequence has been changed in such a way that the enzyme used is no longer able to work. Detection of the various fragments is carried out by means of gel-electrophoresis. That information has been taken essentially from the publication by R.M. Bertina et al., Mutation in blood coagulation factor V associated with resistance to activated protein C, Nature, 369, 1994, 64-67. I-LIj

Claims (12)

1. A method for the detection of mutations of DNAs, in which a DNA is amplified by means of PCR and the amplikons are cleaved and detected with one or more restriction enzymes, two labelled primers being used and the label of the first primers serving to bind the amplikons to one or more supports and the label of the second primers serving for detection, characterised in that supports are used to which supports the first primers can be bound also under the conditions of PCR, and first the first primers are bound to the supports and then amplification of the DNA is carried out with the second primers.

2. A method according to claim 1, characterised in that the supports have an activated surface.

3. A method according to either one of the preceding claims, characterised in that the first primers are partially or com pletely fixed to the supports before the PCR, and the second primers are present in the reaction solution optionally in free form.

4. A method according to any one of the preceding claims, characterised in that one or more restriction enzymes are added before, during or after binding of the amplikons to the sup ports.

5. A method according to any one of the preceding claims, characterised in that the restriction enzymes are incubated with the amplikons. - 13

6. A method according to any one of the preceding claims, characterised in that, after cleavage with the restriction enzymes, washing is optionally carried out and the labels of the second primers are detected.

7. A method according to any one of the preceding claims, characterised in that the detection method is chosen according to the label of the second primers.

8. A method according to any one of the preceding claims, characterised in that the detection can be carried out directly or indirectly according to the label of the second primers.

9. A kit for carrying out the method according to any one of the preceding claims, characterised in that it comprises two labelled primers, one of which is suitable for binding PCR reaction products to one or more supports and the second primer serves for detection.

10. A kit according to claim 9, characterised in that it addi tionally comprises free nucleotides, a polymerase and at least one restriction enzyme.

11. A kit according to any one of the preceding claims, char acterised in that it comprises a suitable support, preferably having an activated surface.

12. The use of a kit according to any one of claims 9 to 11 for the detection of mutations.