RU2193069C2 - Method of detection of mutations of deoxyribonucleic acids using restrictases and kit for assay carrying out - Google Patents

Abstract

FIELD: genetic engineering, biochemistry. SUBSTANCE: invention relates to method of detection of mutations in DNA using restrictases. DNA segment in mutated genome subjected for analysis is copied by polymerase chain reaction. Labeled primers are used. The first label is used for binding DNA segment with carrier surface and the second label is used for detection of mutation. The selected restrictase is incubated with amplicon obtaining after polymerase chain reaction carrying out. Mutated segment is not cleaved if cleavage site is fallen out as result of mutation. After binding segment with carrier surface detection is performed and label of the second primer is removed in the case of DNA of "wild type" but it can be found in mutant. Method ensures to use a lot of labels that can detect mutation directly or indirectly. Invention proposes rapidly acting method for detection of definite genome mutations. EFFECT: enhanced effectiveness of method, decreased labor intensity. 12 cl, 11 dwg, 1 ex

Description

 The invention relates to a method for detecting mutations of deoxyribonucleic acids (DNA), wherein one DNA is amplified by polymerase chain reaction (PCR), and the resulting amplicons are cut by one or more restriction enzymes (restrictase enzymes) and are detected, the method is characterized in that two labeled seeds, the label of the first seeds used to bind amplicons with one or more carriers, and the label of the second seeds used to detect. The invention further relates to a kit for carrying out the above method, characterized in that it comprises two labeled seeds.

 In the process of identifying the human genome, the importance of defective genes in the occurrence of many diseases is shown. So, for example, with many types of cancer, with diseases such as cystic fibrosis, Alzheimer's disease, as well as with the development of thrombosis, changes in genetic information are observed, which in certain cases cause the occurrence of these diseases. Therefore, in medical diagnostics, the detection of such mutations is becoming increasingly important.

 Currently, mutations are often detected using the so-called restriction analysis. In this case, restrictase enzymes are used that are able to recognize double-stranded DNA by its specific sequences and cleave it in the corresponding site. Currently, about 500 different restriction enzymes with more than 100 cleavage sites characteristic of them have been identified. This high specificity is used to detect defects, because a mutation that causes a painful change in the body often enters the boundaries of such an enzyme cleavage site. Due to the mutation, the site changes so much that the chain under investigation cannot be cleaved. In other cases, a site capable of being cleaved by this enzyme appears only as a result of a mutation that did not previously occur.

 To conduct a standard analysis, the genomic DNA under investigation is subjected to PCR, with the help of which it is possible to copy the desired portion of the DNA within which the mutation is located. For this purpose, use the so-called primers - DNA sections - (seed), which determine the beginning and end of the amplified DNA section. Further, for analysis, free nucleotides are also needed - DNA fragments and polymerase - an enzyme that catalyzes the formation of the studied and copied DNA segments. Further, for conducting PCR, they also use a device, a thermostat, which provides the desired temperature regime of PCR.

 After copying the test segment, it is digested with the appropriate restriction enzyme. The latter, under certain conditions, after a mutation cleaves a specific DNA sequence or DNA cleavage does not occur. Depending on the concentration of the enzyme, this process can take several hours.

 Until now, agarose gel electrophoresis has not been used to analyze the product. To do this, polymegarose is dissolved in a buffer solution by heating, and after cooling, the gel-like polysaccharide is poured into an electrophoresis device. After the gel is completely cooled, samples are applied to it and, when a potential difference is applied, DNA fragments begin to move in the agarose gel, and the degree of movement depends on their size.

 DNA fragments can be visualized, for example, by adding fluorescent intercalates, i.e. substances that are introduced into DNA fragments and become visible under the influence of UV radiation. A mutation can be diagnosed when a specific change in behavior relative to a restricted sample is observed in the gel.

 To detect mutations, along with copying the DNA fragment of interest, complex gel electrophoretic methods associated with the preparation of the corresponding gels, application of samples and waiting periods between individual gel electrophoresis operations are used.

 These methods are time consuming, they are complex and, therefore, expensive.

 The objective of the invention is to develop a method and an appropriate kit for its implementation, with which it is possible to detect the above mutations simply and with a high degree of reproducibility and passage of samples, avoiding the disadvantages of gel electrophoresis.

 The problem is solved by a method for detecting mutations of deoxyribonucleic acids (DNA), moreover, one DNA is amplified by PCR, and the resulting amplicons are cut with one or more restriction enzymes and are detected, i.e. are analyzed, characterized in that they use two labeled seeds, with the label of the first seeds used to bind amplicons (copied DNA fragments) with one or more carriers, and the label of the second seeds used for detection (analysis).

 The PCR used in the implementation of the present method is carried out in the usual way, and the seeds of the type described above are used as seeds.

 According to the invention, preferably, carriers are used which, under PCR conditions, also allow the binding of the first seeds. In this case, carriers having an activated surface, such as a suitably treated surface of a reaction vessel or a microtiter plate, turned out to be particularly preferred.

 According to the proposed method, the first seeds are partially or completely fixed on the media either before, during, or after PCR, while the second seeds are preferably present in the reaction solution in a free, i.e., not fixed on the media form and, if necessary, excess amount. Seeds can be added to the reaction solution either simultaneously or sequentially.

 The second seeds should preferably have labels that are easily detectable by conventional methods.

 According to the invention, before, during or after binding of the DNA segments (amplicons obtained by PCR), one or more restriction enzymes can be added to the carriers, preferably after the binding process. According to the invention, restriction enzymes can be incubated with amplicons.

 As a restriction enzyme, all known restriction enzymes can be used, with which enzyme or enzymes to be used in a particular case, of course, depending on the cleavage sequence.

 After fixing the amplicons on suitable carriers with the first seeds and before and / or after splitting, it is preferable, however, after splitting the amplicon with one or more of the above restriction enzymes, the amplicons are washed with a suitable washing agent, for example, if necessary, with deionized or distilled water containing, if necessary the need for additives, such as buffers. When washing is carried out after cleavage, cleaved but not fixed DNA segments are removed.

 According to the invention, the detection methods are chosen so that it is possible to detect the label of the second seeds. In this case, the detection process in accordance with the type of labels of the second seeds can be carried out directly or indirectly.

 In the practice of biochemical analysis, many detection methods are known.

 1. Conjugation with substances directly detected by spectroscopy.

 2. The use of enzymatic labels, which turn their substrate into photochemically detectable substances. An advantage of the latter method is the amplification of the associated measurement signal.

 According to a first embodiment of the invention (see Scheme 1), the DNA is first amplified and the resulting amplicons bind to a suitable carrier, with the label on the first seed serving as a binding factor. If necessary, the first seeds may also include several such labels.

 According to a second embodiment of the invention (see Scheme 2), the first seeds are first bound to an appropriate carrier, for example, to the surface of a vessel in which PCR proceeds. In this case, the chosen pairing method should satisfy the PCR boundary conditions, for example, heat resistance. The reaction then proceeds under standard conditions, wherein the second seed is not bound to the carrier and is preferably in excess relative to the first seed.

 During PCR, the desired DNA segment forms in the solution, and the hybridization and amplification processes initiated by the already bound seed also occur on the surface of the carrier. At the end of PCR, the desired products are obtained that are directly linked to the carrier. Then, the excess material necessary for PCR (e.g., free nucleotides) and unbound reaction products are removed by washing. The segment is then digested with the appropriate restriction enzyme, if necessary, another washing step is carried out, and finally, the above detection is carried out.

 According to scheme 1, it is possible to label, for example, biotin. At stage 3, according to this scheme, pairing with the carrier is carried out, for example, by means of a microtiter plate through a biotin-binding protein, for example streptavidin or avidin, which is adsorbed on the surface of the carrier or covalently bound, for example, on the surface of the wells of a microtiter plate. Microtiter tablets coated with a streptavidin layer are commercially available (form Beringer, Mannheim).

 According to Scheme 2, for seed 1, covalent conjugation with a carrier, for example a microtiter plate, can be provided. For this purpose, for example, phosphorylated seeds can be used, which, as part of the carbodiimide reaction, can react with carriers which are activated accordingly. An example of suitable functional groups of a carrier, for example a microtiter plate, are amino groups. Mirk-titration tablets of this kind are also commercially available.

 In the case of Schemes 1 and 2, the same labels can be used for seeds 2. Suitable examples are fluorescein and its derivatives, rhodamine and its derivatives, Cy5 (fluorescent dye), Cy3 (fluorescent dye) or compounds that are indirectly detected.

 - Fluorescent dyes: in the case of these seeds, direct subsequent detection with a suitable fluorimeter is possible.

 - Digoxin: indirect detection is possible after the addition of an enzyme-labeled antibody.

 - Biotin: indirect detection is possible after the addition of an avidin conjugate and an enzyme, for example, after the addition of avidin or streptavidin.

 The fact of binding of the corresponding recognition protein (antibodies, avidin or streptavidin) as in other assays with microtiter plates can be detected by adding suitable enzyme substrates, in particular peroxidase or alkaline phosphatase enzymes (moreover, colored products are obtained by reaction with the enzyme), or directly, if the pairing process is carried out not on microtiter tablets, but on suitable detectors. In this case, suitable detectors can be, for example, planar waveguides known from the biosensor technique, on which conjugation reactions can be carried out using the seed 1 described above for microtiter plates.

 According to the invention, the studied DNA, which is a wild type or mutant, may have a restriction site.

a) The presence of a restriction site in intact wild-type DNA:
In the event of a mutation in the DNA region of interest, the restriction site of the DNA is deleted in accordance with the selected example. Moreover, to prevent specific recognition and cleavage of the segment by an enzyme, a point mutation is sufficient. In the subsequent washing step, prior to the actual detection, the restriction enzyme, PCR starting products and, if necessary, PCR products containing the detectable label can be removed.

 At the detection stage, the presence or absence of the label is analyzed. In the case of a DNA mutation, a detectable label occurs, and an appropriate reaction is observed depending on the type of label selected. In the case of a label with an enzyme, for example, a catalyzed color reaction is observed. In the absence of mutation, the enzyme is capable of specific cleavage and the label is removed, so that the detected signal is absent.

b) The presence of a restriction site in mutated DNA:
In the event of a mutation in the DNA region of interest, a DNA restriction site is created in accordance with the selected example. Moreover, for specific recognition and cleavage of a segment by an enzyme, a point mutation is sufficient. In the subsequent washing step, prior to the actual detection, the restriction enzyme, PCR starting products and, if necessary, isolated PCR products containing the detectable label can be removed.

 At the detection stage, the presence or absence of the label is analyzed. In the case of a DNA mutation, a detectable label is absent and no reaction occurs. In the absence of mutation, the enzyme is not capable of specific cleavage, and the label is not removed, and therefore, a detectable signal is obtained.

The invention is explained in detail below with drawings illustrating the above case a):
Scheme 1 illustrates the first sequence of steps of the proposed detection method. Figure 1 shows the amplification of the target using labeled seeds. PCR is carried out in suitable vessels. Figure 2 shows labeled PCR products. One end of the PCR products has a label that serves for subsequent binding to the surface of the carrier, and a label at the other end serves to detect. The desired sequence containing a possible mutation is within the limits of the amplified segments.

 Figure 3 shows the process of linking segments due to the first label; in FIG. 4 depicts the process of exposure to a restriction enzyme in order to determine whether or not a restriction site has been deleted due to a mutation. In the case of a mutation, as shown in figure 5, the restriction site is absent and the detected labels are not deleted. Conversely, in the absence of mutation, the enzyme is capable of cleavage. A simple flushing can separate the segment, so that the connected part of the segment does not give a signal.

 Scheme 2 illustrates the second sequence of steps of the proposed detection method. Figure 1 shows that part of the first seed, which serves to bind to the surface of the carrier, is in a bound form even before the start of PCR. The second seed that can be detected is in excess in solution. Figure 2 shows that at the end of the PCR amplicon containing the test sequence is fixed on the surface of the carrier. In FIG. 3 depicts the addition of a restrictase, which, as described above, is capable of cleaving a wild-type sequence, but not a mutated sequence. In FIG. 4 shows the result of the detection. After the (not shown) washing step, a detectable label in the case of a mutation is still on the fixed segment. However, in the case of the wild-type, no signal is obtained, since, due to the presence of the restriction site, the restriction enzyme can cleave the label-bearing segment, which is removed by washing after separation.

 The invention also relates to a kit for carrying out the proposed method, characterized in that it includes two labeled seeds and, in addition, preferably contains additional free nucleotides and one polymerase.

 In this case, the first seed serves to bind PCR products to one or more carriers, and the second to detect mutations, and the used carrier preferably has an activated surface.

 Compared with the prior art, the proposed method and kit have the following advantages.

 - The described forms of carrying out the invention can save time. The second embodiment of the invention has particular advantages, since both PCR and the analysis of its products are carried out in one vessel. Due to this, there is no need to transfer samples with a pipette from one vessel to another, which simplifies handling and eliminates the occurrence of errors associated with such operations.

 - Depending on the type of label detected, a mutation is detected immediately after digestion with a restriction enzyme, and not at the end of gel electrophoresis.

 - In clinical practice, testing is often associated with a large number of samples. Therefore, the process of processing samples using a known method for detecting mutations, based on gel electrophoresis, is very laborious. Since the proposed method can be carried out completely on microtiter tablets, it has the advantage of simultaneously processing, for example, 96 (or 384) samples.

 - In the case of gel electrophoresis, one has to work with toxic DNA intercalation compounds (intercalates). Using the proposed method, you can completely abandon the use of these compounds.

 Example 1

 A mutation in human factor V is considered to be responsible for increasing the risk of thrombosis, since it inhibits the breakdown of factor V glycoprotein by ABS serine protease (activated protein C). Human factor V is a glycoprotein with a molecular weight of 330 kDa, which interacts specifically with ABS and, as a rule, is cleaved by it in a certain sequence site - in the case of a human protein, after Arg 506 (arginine is the 506th amino acid of the protein). When examining the sequence of the corresponding gene, it was found that when at site 1, 691 of the nucleotide sequence G (guanine) is replaced by A (adenine), there is an increased risk of thrombosis. This mutation leads to the fact that in the protein Arg 506 (the product of the nucleotide sequence of CGA) is replaced by glutamine (Gln) (the product of the nucleotide sequence of CAA) and, therefore, the above protease cleavage no longer occurs. In order to detect this mutation, seeds are used, with which it is possible to copy a portion of the gene, including the corresponding nucleotide sequence, within the framework of PCR. The result is, for example, fragments with a length of 267 base pairs. Then, in the case of the presence of guanine in site 1, 691 by treatment with restriction enzyme MnlI, three fragments of 67, 37 and 163 base pairs in length are obtained, respectively, since the enzyme, due to its sequence specificity, is capable of cleaving a gene fragment at two sites (after 67 and after after 37 base pairs ) In the case of a mutation to adenine, the second cleavage site disappears. As a result, only two cleavage products are observed with a length of 67 and 200 base pairs, respectively, since due to the mutation the sequence is so changed that the enzyme used can no longer act. Detection of individual gene fragments is carried out by gel electrophoresis. This information is mainly known from a 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.

Claims (12)

 1. A method for detecting mutations of deoxyribonucleic acids (DNA), in which one DNA is amplified by polymerase chain reaction (PCR), and the resulting amplicons are cut with one or more restrictase enzymes and detected, using two labeled seeds and the label of the first seeds serves to bind amplicons with one or more carriers, and the label of the second seeds serves to detect, characterized in that they use carriers that can bind the first seeds also in PCR conditions, and first bind The first seeds with carriers and then using the second seeds amplify the DNA.  2. The method according to p. 1, characterized in that the carriers have an activated surface.  3. The method according to one of the preceding paragraphs, characterized in that the first seeds before PCR are partially or completely associated with carriers, and the second seeds, if necessary, are in the reaction solution in free form.  4. The method according to one of the preceding paragraphs, characterized in that one or more restriction enzymes are added before, during or after binding of the amplicons to carriers.  5. The method according to one of the preceding paragraphs, characterized in that the restriction enzymes are incubated with amplicons.  6. The method according to one of the preceding paragraphs, characterized in that after cleavage with restrictase, if necessary, carry out washing and detect the labels of the second seeds.  7. The method according to one of the preceding paragraphs, characterized in that the detection method is selected in accordance with the label of the second seeds.  8. The method according to one of the preceding paragraphs, characterized in that the detection is carried out directly or indirectly in accordance with the label of the second seeds.  9. A kit for carrying out the method according to one of the preceding paragraphs, the kit includes two labeled seeds, characterized in that one of the seeds for binding amplicons is associated with one or more solid carriers, and the second serves for detection, and amplification is performed with the second seed DNA  10. The kit according to p. 9, characterized in that it further includes free nucleotides, one polymerase and at least one restrictase.  11. Set according to one of the preceding paragraphs. 9 and 10, characterized in that it includes a suitable carrier, preferably with an activated surface.  12. Set according to one of paragraphs. 9-11, characterized in that it is suitable for detecting mutations.

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