RU2688189C1 - Oligonucleotide sequence oligonucleotide sequence for the nras gene q61r mutation in the tumor formations of the thyroid gland - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: proposed group of inventions relates to medicine. Disclosed are a method and a kit for detecting Q61R mutation in a NRAS protein in a human tumor tissue sample. Method involves obtaining a sample of biological material taken from a patient, recovering DNA from said sample and carrying out PCR using a kit for detecting Q61R mutation in a NRAS protein.

EFFECT: presented group of inventions enables higher specificity and sensitivity of determining the presence of a mutant allele at the Q61R point of the NRAS gene, as well as reducing the probability of process errors during laboratory manipulations.

3 cl, 1 dwg, 1 tbl, 1 ex

Description

Technical field

The invention relates to the field of medicine, namely to genetics, oncology, molecular diagnostics, and relates to a method for detecting the Q61R gene mutation in the NRAS gene in human tumor tissues and can be used to quickly search for a mutation in the analyzed point.

The level of technology

In many oncological diseases, in particular in thyroid cancer, the NRAS gene is an indispensable target for the detection of gene mutations. NRAS is an oncogene whose mutations have the potential to transform normal cells into malignant ones by activating signaling pathways in cells. Mutations in the NRAS gene are often a marker for aggressive thyroid cancer behavior and poor prognosis. Mutations in the NRAS gene, which can cause unlimited cell growth, affect a single amino acid in the NRAS protein. In particular, they replace amino acid glutamine at position 61 with arginine (Q61R), which leads to the formation of a permanently active protein. Instead of triggering cell growth in response to certain signals from the outside, the overactive protein constantly directs cells to grow and divide. The mutational status of the NRAS gene is related to drug resistance, especially for patients receiving tyrosine kinase inhibitors. Detection of this mutation allows targeted clinical therapy of oncological patients. This will allow to choose the most promising treatment protocol individually, based on the molecular genetic profile of the patient’s tumor. This mutation is characteristic for the development of adenocarcinoma in colorectal cancer, occurs with a frequency of about 5%, with urothelial carcinoma and bladder cancer - about 2%, with hepatocellular carcinoma - 4%, in the case of adenocarcinoma of lung cancer - about 1%, with large cell lung cancer - 4%, and with melanoma, the frequency of occurrence of this mutation is about 20%.

The classic method for determining mutations is Singer sequencing. The disadvantages of the method when used in the clinic: low sensitivity, complexity, risk of cross-contamination of clinical samples. In addition, for further decoding of chromatograms, highly qualified personnel is necessary, which prevents the use of this method in large-scale studies.

There is another method for detecting NRAS gene mutation by DNA melting (DNA Melting Analysis), which is implemented in high resolution mode (High Resolution Melting Analysis, HRMA) (Erali and Wittwer, Methods, 2010, 50, 250- 261). The disadvantages of the method: relatively low sensitivity, the need for expensive equipment.

There is a method for detecting gene mutations using peptide nucleic acids in the PCR process that can selectively suppress the amplification of wild-type alleles and thereby increase the sensitivity of mutation detection (Guha et al., DISSECT Method Using PNA-LNA Mutation. PLoS ONE, 2013, 8, e67782). The disadvantages of the method: the need for independent identification of the mutant allele, costly chemical synthesis.

The method of hybridization on microchips is the most promising for carrying out simultaneous genotyping of a large number of genetic loci, however, at the moment, there are no cheap test systems based on this method (Emelyanova et al., Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1 / 2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis Oncotarget, 2017 Apr 10; 8 (32): 52304-52320).

The method of setting allele-specific PCR is quite common, but requires the setting of independent parallel PCR reactions. Evaluation of results also requires the formulation of gel electrophoresis in polyacrylamide gel, which dramatically reduces its value for large-scale analysis of a large number of samples of tumor tissue of patients.

At the moment there are only a small number of patents in which the NRAS gene would appear and its association with the development of thyroid tumors, one of such patents is WO 2017117523 A1, in which the authors suggest using the sequencing method of the new generation (NGS) to analyze the panel of genes involved in the development of thyroid tumors. However, this method is quite expensive.

Thus, at the moment there is an urgent need to develop a method for diagnosing a Q61R mutation in the NRAS gene, which would compare favorably with the known solutions by simplicity of analysis, availability and low cost. Since all currently available methods for determining the Q61R mutation in the NRAS gene have some disadvantages, there is a real need to create a simple, inexpensive and specific method for identifying this mutation, with a view to further introduction into any standard clinical laboratory.

This method is provided by the present invention.

Summary of Invention

The task of the claimed invention is to create a new, more sensitive method for detecting the Q61R mutation of the NRAS gene in human tumor tissues using the method of real-time PCR.

The definition of the studied alleles at the indicated point of the NRAS gene is achieved by using a pair of fluorescently-labeled TaqMan probes, which allow analysis by the level of accumulation of the fluorescent product of each of the probes during the PCR reaction.

The inventive method has a high sensitivity to detect the Q61R gene mutation of the NRAS gene, while maintaining the simplicity of execution, implementation of a 2-hour test in one tube without intermediate and additional procedures with a single recording of the results and without the risk of cross-contamination of DNA samples of tumor tissues enclosed in paraffin blocks.

The essence of the proposed method consists in detecting the Q61R mutation of the NRAS gene in the tumor DNA using real-time PCR with two TaqMan fluorescent probes. The presence of such a probe is a feature of real-time PCR - in addition to the possibility of conducting PCR with visualization of results in real-time, the specificity of the reaction is also enhanced by the probe as compared to conventional PCR.

This problem is solved by creating a kit for detecting a Q61R mutation in the NRAS protein in a sample of human tumor tissue, where the kit includes oligonucleotides having the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO : 4, wherein each of the oligonucleotides with the sequences SEQ ID NO: 1 and SEQ ID NO: 2 contains at one of the ends a fluorescent dye selected from the group FAM or VIC, attached by means of a covalent bond, and at the opposite end a fluorescence quencher selected from the group BHQ1 or BHQ2, attached a covalent bond, and wherein the oligonucleotides SEQ ID NO: 1 and SEQ ID NO: 2 have different dyes and fluorescence quenchers.

In some embodiments of the invention, this kit is characterized by the fact that it further includes a DNA polymerase enzyme, a reaction buffer, and a mixture of four different deoxyribonucleotides.

This task is also solved by creating a method for detecting a Q61R mutation in an NRAS protein in a patient, comprising at least the following stages: (a) a sample of biological material taken from the patient is obtained; (b) DNA is isolated from the indicated sample, wherein the DNA contains the NRAS gene; (c) carry out a PCR reaction using the above kit; (d) when a signal is detected in this PCR reaction from a dye bound to the oligonucleotide of SEQ ID NO: 1, the presence of a mutation Q61R in the NRAS protein in the sample of the specified patient is detected, and when a signal from the dye bound to the oligonucleotide SEQ ID NO: 2 is detected, state the absence of a Q61R mutation in the NRAS protein in a sample of the specified patient.

The technical result of the present invention is to increase the degree of specificity and sensitivity of the diagnostic test system designed to determine the presence of a mutant allele at the Q61R point of the NRAS gene, as well as reducing the time for diagnosis while reducing the likelihood of technological errors during laboratory manipulations due to the convenient format of the proposed test system .

Brief description of drawings

Fig. 1. Accumulation of fluorescence of TaqMan probes during PCR diagnostics - amplification of the NRAS gene. (A) Graph of fluorescence signal accumulation upon amplification of a specific pathogen nucleic acid fragment, FAM detection channel; (B) graph of fluorescence signal accumulation, VIC detection channel.

Detailed disclosure of the invention

In the description of this invention, the terms "includes" and "including" are interpreted to mean "includes, among other things." These terms are not intended to be interpreted as “consists only of”. Unless otherwise defined, technical and scientific terms in this application have standard meanings commonly accepted in scientific and technical literature.

The term “NRAS” or “human NRAS” refers to the gene encoding the small GTPase (G-protein, membrane-bound protein involved in signal transmission), the sequence of which is listed in the National Center for Biotechnology Information (NCBI) under the number Gene ID_4893 (https: //www.ncbi.nlm.nih.gov/gene/4893), as well as to allelic variants of this gene (isoforms) present in the genomes of patients. The unique identifier of this gene according to the Human Genome Nomenclature Committee of the European Bioinformatics Institute (HGNC): 7989. Attached to the inner side of the cell membrane, RAS proteins are the first members of the signaling cascade that lead to the activation of gene transcription and cell proliferation.

The main objective of the present invention is to develop a simplified and affordable method of analyzing genetic polymorphism at point Q61R for conducting clinical DNA diagnostics of patient's tumor tissues. The proposed method has high specificity and sensitivity in the analysis of DNA isolated from paraffin blocks of patients. This test system is suitable for the rapid determination of the presence or absence of a point mutation Q61R in the NRAS gene. As clinical samples, DNA samples isolated from paraffin blocks obtained from the patient being examined can be used. The DNA sample serves as a matrix for the further implementation of the method for detecting mutations.

To obtain specific PCR products, oligonucleotides that differ in specificity for the analyzed section of the NRAS gene were selected.

They were developed according to the following criteria:

- absence of internal secondary structure;

- lack of complementarity between the 3'-ends, which may be the reason for the formation of primer dimers;

- the presence of a single melting point (the range of melting points of the primers is not more than 1 ° C of the average value);

- lack of complementarity of primer sequences for the 1st round with sequences of other genes in the human genome;

- the ability to amplify gene fragments containing areas that allow to identify further point mutation Q61R in the NRAS gene.

The basis of the disclosed technical solution is the creation of an optimal set of oligonucleotide sequences for determining the Q61R mutation of the NRAS gene having the following nucleotide composition: TACTCTTCTCGTCCAGCTGT (SEQ ID NO: 1) and TACTCTTCTGTCCAGCTGT (SEQ ID NO: 2). At the same time, these oligonucleotides serve as the basis for TaqMan probes used in real-time PCR reactions. The implemented version of the probes is shown below, while in other embodiments, the FAM and VIC dyes can be used both with one and with another oligonucleotide:

FAM - TACTCTTCTTGTCCAGCTGT - BHQ1 (TaqMan probe for non-mutated variant allele at the analyzed point), and

VIC - TACTCTTCTCGTCCAGCTGT - BHQ2 (TaqMan probe for mutant variant of allele at the analyzed point).

One of the most common channels for the detection of fluorescence, at the moment, are the FAM and VIC channels. This is the most suitable pair of probes that are ideally suited to each other in their spectral and chemical properties. The FAM channel has a wavelength in the range of 492-516 nm., And the VIC channel has a 535-555 nm. Thus, both probes for real-time PCR are oligonucleotides to which are attached fluorophore molecules (carboxyfluorescein (FAM / VIC)) and fluorescence quencher molecules (Black Hole Quenchers (BHQ1 / BHQ2)). In this case, FAM or VIC can be used for each of the probes, and the necessary condition is the attachment of different dyes to each of the two probes. That is, if a VIC is attached to SEQ ID NO: 1, then FAM must be attached to SEQ ID NO: 2, and vice versa. Similarly, BHQ1 or BHQ2 can be used for each of the probes. In some embodiments, it is allowed to use a single absorber (BHQ1) for both probes.

The primer sequences that accumulate the region of interest of the NRAS gene with the analyzed Q61R mutation in the PCR reaction have the following sequences:

direct primer: 5'-CCACACCCCCAGGATTCTTAC-3 '(SEQ ID NO: 3), and

reverse primer: 5'-CGCCTGTCCTCATGTATTGG-3 '(SEQ ID NO: 4).

Thus, when carrying out the PCR reaction with the participation of SEQ ID NO: 3 and SEQ ID NO: 4, the following sequence is generated from the NRAS gene (the mutation of interest is shown in parentheses):

To ensure a balanced and effective amplification of the studied samples, such parameters as the concentration of magnesium chloride and PCR primers in the mixture, the number of amplification cycles, the time of elongation, annealing, and denaturation should also be optimized.

The inventive primer set is used as follows:

1. Obtaining biological material from the patient. As a source of DNA from a patient, paraffin FFPE blocks, biopsies after a fine-needle biopsy, and samples of cells from cytological glasses can be used.

2. DNA isolation from FFPE blocks. Fast and high quality DNA extraction can be performed using commercially available reagent kits, for example, QIAamp DNA FFPE Tissue Kit, QIAGEN (https://www.qiagen.com/us/shop/sample-technologies/dna/genomic-dna/qiaamp- dna-ffpe-tissue-kit). DNA extraction from biopsy samples was carried out using RealBest DNA kits - extraction 2, RealBest DNA express, RealBest extraction 100, BEST Vector (http://vector-best.ru/prod/index.php?pcat=pcr&gid=181), DNA isolation from cytological glasses was carried out using a set of Pinpoint Slide DNA Isolation System, a company (Zymo Research https://www.zymoresearch.eu/pinpoint-slide-dna-isolation-system). Also, other similar DNA extraction kits can be used.

3. Setting PCR in real time. Amplification should be performed using the proposed set of primers and TaqMan probes specific to the study area at the analyzed point Q61R.

4. Analysis of PCR results in real time due to changes in the dynamics of the accumulation of the fluorescent reaction product for each of the TaqMan probes.

For PCR, ready-made commercially available kits can be used, containing all the necessary components, with the exception of primers and TaqMan probes. To detect the mutation of the NRAS gene in the DNA of a tumor, it is necessary to find out which channel signal is detected by the PCR machine. If a non-mutant variant of the gene is present in the sample, the device detects a signal from the FAM channel. This indicates the presence of a “wild”, non-mutant allele in the analyzed point mutation - the presence of nucleotide A. The signal from the VIC channel is detected by the instrument in all cases when a mutant variant is present in the sample (nucleotide G).

The following examples of the method are given in order to disclose the characteristics of the present invention and should not be construed as in any way limiting the scope of the invention.

Example. Detection of the Q61R mutation of the NRAS gene in patient samples.

To demonstrate the possibility of using this kit, molecular diagnostics of the NRAS gene was performed in samples of 11 patients who were diagnosed with a thyroid tumor and underwent surgery to remove masses, during which pieces of tumor tissues were promptly fixed with formalin and embedded in paraffin. All patients who were assigned to this analysis were sent from the National Medical Research Center (FGBU "NMITS Endocrinology" of the Ministry of Health of Russia).

For molecular genetic studies, biological material was DNA isolated from paraffinic FFPE blocks of patients. The use of these blocks is extremely convenient for the researcher, since allows you to store fixed tumor tissue of the patient at a temperature of + 4 ° C for several years. In this case, it is expressed in the fact that there is an opportunity to take into the analysis of tissue that was obtained during the operation, which took place some time before the analysis. After surgery, FFPE blocks are handed out to the patient and stored with him. The patient can use the FFPE block at any time and provide his surgical material when the need arises. DNA from the blocks was isolated by standard protocols provided by manufacturers of DNA extraction kits from FFPE blocks. The analysis was taken only that piece of tissue, which was directly a tumor. This was possible due to the fact that the histologist, looking through the histological glasses of each block, made the lining of the material, indicating the boundaries of the tumor. Paraffin blocks were cut using a microtome - 8 shavings of 10 microns each. The material for PCR was precisely the tumor DNA from the FFPE block.

The amplification reaction was performed using special equipment - DT-Prime M1 amplifier (firm "DNA Technology") according to the following protocol for preparing the reaction mixture for PCR:

- 2.5 μl of deoxyribonucleotide triphosphates (25 mM / μl);

- 2.5 μl MgCl ₂ (25 mM / μl);

- 2.5 μl of 10X buffer (100 mM Tris-HCl (pH 8.8 at 25 ° C), 500 mM KCl, 0.8% P40, 20 mM MgCl ₂ );

- 0.5 µl Hot-Start Tag Polymerase (50X);

- direct primer (10 pmol / μl) - 0.5 μl per reaction;

- reverse primer (10 pmol / μl) - 0.5 μl per reaction;

- probe-specific oligonucleotide, per non-mutant allele (10 pmol / µl) - 0.5 µl per reaction;

- probe-specific oligonucleotide, per mutant allele (10 pmol / µl) - 0.5 µl per reaction;

- DNA (33 ng / μl) - 3 μl per reaction;

- water.

The program used for the amplification had the following temperature regimes:

95 ° C - 2 min.

95 ° C - 15 sec.

58 ° C - 15 sec. 40 cycles

72 ° С - 20 sec. (we put the detecting label)

72 ° C - 5 min.

Storage (+ 4 ° С)

The results were evaluated by comparing the values of the fluorescence onset cycle (Ct) for the control and test samples under the condition of amplification efficiency of 95-100% and by analyzing the standard curve (Fig. 1A, B).

The determination of the presence of a non-mutant or mutant allele at the analyzed point Q61R is related to which channel detects the device in the sample. If the FAM channel is detected in the sample, then there is a non-mutant allele in the analyzed sample. Under the condition that the device detects a signal from the VIC channel, a mutant allele was detected in the patient's DNA sample under study. As can be seen from table 1, all samples, except 11, show a signal from the FAM detection channel, as well as K- (negative control); therefore, there is no mutation at these points in Q61R in these patient DNA samples. In sample 11 and in the positive control (K +), the system detected a signal from the VIC channel; therefore, in this case, there is a mutant allele in the analyzed samples. The results of the amplification of all samples that were analyzed by this method were confirmed by the method of sequencing the selected DNA by Sanger.

With the indicated amplification parameters and the composition of the reaction mixture, the numerical values presented in Table 1 were obtained. These values characterize the minimum cycles at which the onset of fluorescence is recorded.

Thus, the developed universal set of primers and probes, as well as real-time PCR reaction conditions adapted for their use, make it possible to increase the efficiency and accuracy of identification of the mutant and non-mutant allele in the Q61R point of the NRAS gene. The application of this invention allows to optimize molecular genetic diagnosis in tumors of the thyroid gland, as well as in samples of other human tumors. Low cost and simplicity of the study allow to use this method of detection in almost any PCR laboratory.

Although the invention has been described with reference to the disclosed embodiments, it should be obvious to those skilled in the art that the specific details of the cases described are given only to illustrate the present invention and should not be construed as limiting the scope of the invention in any way. It should be clear that it is possible to implement various modifications without departing from the gist of the present invention.

Claims (7)

1. A kit for detecting a Q61R mutation in an NRAS protein in a sample of human tumor tissue, wherein said kit includes oligonucleotides having the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, each of the oligonucleotides with the sequences SEQ ID NO: 1 and SEQ ID NO: 2 contains at one end a fluorescent dye selected from the FAM or VIC group, attached by means of a covalent bond, and at the opposite end, a fluorescence quencher selected from the BHQ1 group or BHQ2, attached by means of a covalent bond, and onukleotidy SEQ ID NO: 1 and SEQ ID NO: 2 have different dyes and fluorescence quenchers. 2. The kit according to claim 1, further comprising a DNA polymerase enzyme, a reaction buffer, and a mixture of four different deoxyribonucleotides. 3. A method for detecting a Q61R mutation in an NRAS protein in a patient, comprising at least the following steps: (a) receive a sample of biological material taken from the patient; (b) DNA is isolated from the indicated sample, wherein the DNA contains the NRAS gene; (c) conduct a PCR reaction using the kit of claim 1; (d) when a signal is detected in this PCR reaction from a dye bound to the oligonucleotide of SEQ ID NO: 1, the presence of a mutation Q61R in the NRAS protein in the sample of the specified patient is detected, and when a signal from the dye bound to the oligonucleotide SEQ ID NO: 2 is detected, state the absence of a Q61R mutation in the NRAS protein in a sample of the specified patient.

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