RU2674687C1 - Method for analysis of somatic mutations in gnaq and gna11 genes using lna-blocking multiplex pcr and subsequent hybridization with oligonucleotide biological microchip (biochip) - Google Patents

Abstract

FIELD: biology; medicine.SUBSTANCE: invention relates to the field of genetics, molecular biology and medicine. Method for detecting somatic mutations is proposed Q209P (s. 626A>C), Q209L (s. 626A>T), Q209R (s. 626A>G) in the gene GNAQ and Q209L (s. 626A>T), Q209P (s. 626A>C) in the gene GNA11. Carry out the amplification of fragments of the genes GNAQ and GNA11 using LNA-blocking multiplex "nest" PCR. Biochip is used to identify somatic mutations in the GNAQ and GNA11 genes and hybridization of labeled PCR product on the biochip. Record the results of hybridization.EFFECT: invention provides the creation of an analysis method that allows to detect 5 somatic mutations in a short time and with little material costs.3 cl, 2 dwg, 4 ex

Description

FIELD OF THE INVENTION

The invention relates to the field of genetics, molecular biology and medicine, and relates to a method for determining somatic mutations in the GNAQ and GNA11 genes using the technology of LNA-blocking multiplex PCR and subsequent hybridization with an oligonucleotide biological microchip (biochip). The occurrence of somatic mutations in the GNAQ and GNA11 genes is an early event in the formation of uveal melanoma, which can develop from a pre-existing nevus. Identification of these mutations is important for diagnostic and prognostic purposes. Targeted drugs are currently being developed to treat patients with GNAQ / GNA11 mutations.

State of the art

A large number of methods for determining somatic mutations are known in which various tools are used to analyze the unique nucleotide sequence of human DNA. Conventionally, among them six groups of methods can be distinguished:

- Enzymatic methods:

amplified fragment length polymorphism analysis (AFLP);

restriction fragment length polymorphism analysis (RFLP);

Cleavage Cleavage (CFLP);

resolvase cleavage (EMD); analysis based on ligase reaction (LDR, LCR);

PCR enriched in mutant sequence;

Real-time PNA blocking PCR (PNA-clamp PCR);

Real-time LNA-blocking PCR (LNA-clamp PCR);

PCR with direct synthesis termination (DT-PCR);

allele-specific PCR (AS-PCR, PCR-SSP);

SMart amplification;

Real-time PNA-LNA-blocking PCR (PNA-LNA-clamp PCR); Sanger Sessening;

high throughput sequencing (NGS);

digital drip PCR (ddPCR).

- Chemical methods:

chemical splitting of heteroduplexes;

chemical ligation.

- Methods based on different electrophoretic mobility of polymorphic DNA sections:

single chain fragment conformation analysis (SSCP);

heteroduplex analysis (HA);

separation of amplification products by capillary electrophoresis.

- Solid phase detection:

hybridization on oligonucleotide matrices;

elongation of immobilized primers (minisequencing);

pyrosequencing.

- Chromatographic methods:

high performance liquid chromatography (HPLC).

- Physical methods:

mass spectrometry;

resonance fluorescence quenching (FRET);

luminescence, depending on the local environment;

high resolution melting curve analysis.

The most common methods presented are:

1. Sanger sequencing

GNAQ and GNA11 mutations occur in 9.5% of mucosal melanoma and are associated with poorprognosis / X. Sheng, Y. Kong, Y. Li, Q. Zhang, L. Si, C. Cui, Z. Chi, B. Tang, L Mao, B. Lian, X. Wang, X. Yan, S. Li, J. Dai, J. Guo // Eur J Cancer. - 2016. - Vol. 65. - P. 156-163.

L. Al-Olabi, J. St-Onge, D.J. Atherton, H. Aubert, L. Bagazgoitia, S. Barbarot, E. Bourrat, C. Chiaverini, W.K. Chong, Y. Duffourd, M. Glover, L. Groesser, S. Hadj-Rabia, H. Hamm, R. Happle, I. Mushtaq, J.P. Lacour, R. Waelchli, M. Wobser, P. Vabres, E.E. Pattern, V.A. Kinsler // J Invest Dermatol. - 2016. - Vol. 136(4). - P. 770-778.Mosaic Activating Mutations in GNA11 and GNAQ Are Associated with Phakomatosis Pigmentovascularis and Extensive Dermal Melanocytosis / AC Thomas, Z. Zeng, JB Riviere, R.

L. Al-Olabi, J. St-Onge, DJ Atherton, H. Aubert, L. Bagazgoitia, S. Barbarot, E. Bourrat, C. Chiaverini, WK Chong, Y. Duffourd, M. Glover, L. Groesser, S. Hadj-Rabia, H. Hamm, R. Happle, I. Mushtaq, JP Lacour, R. Waelchli, M. Wobser, P. Vabres, EE Pattern, VA Kinsler // J Invest Dermatol. - 2016. - Vol. 136 (4). - P. 770-778.

2. High performance sequencing (next generation sequencing, NGS)

Targeted next generation sequencing reveals unique mutation profile of primary melanocyte tumors of the central nervous system / J. van de Nes, M. Gessi, A. Sucker, I. Moller, M. Stiller, S. Horn, S.L. Scholz, C. Pischler, N. Stadtler, B. Schilling, L. Zimmer, U. Hillen, R.A. Scolyer, M.E. Buckland, L. Lauriola, T. Pietsch, A. Waha, D. Schadendorf, R. Murali, K.G. Griewank // J Neurooncol. - 2016. - Vol. 127 (3). - P. 435-444.

3. Analysis of the conformation of single-stranded DNA fragments (Single-strand conformation polymorphism analysis, SSCP)

Somatic mutation of GNAQ gene is rare in common solid cancers and leukemias / H.S. Eom, M.S. Kim, S.Y. Hur, N. Yoo, S. Lee // Acta Oncologica. - 2009 - Vol. 48. - P. 1082-1084.

GNA11 and N-RAS mutations: alternatives for MAPK pathway activating GNAQ mutations in primary melanocytic tumors of the central nervous system / M. Gessi, J. Hammes, L. Lauriola, E. Dorner, J. Kirfel, G. Kristiansen, A. zur Muehlen, D. Denkhaus, A. Waha, T. Pietsch // Neuropathol Appl Neurobiol. - 2013 .-- Vol. 39 (4). - P. 417-425.

4. Capillary electrophoresis

Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials / C.M. Lovly, K.B. Dahlman, L.E. Fohn, Z. Su, D. Dias-Santagata, D.J. Hicks, D. Hucks, E. Berry, C. Terry, M. Duke, Y. Su, T. Sobolik-Delmaire, A. Richmond, M.C. Kelley, C.L. Vnencak-Jones, A.J. Iafrate, J. Sosman, W. Pao // PLoS One. - 2012. - Vol. 7 (4). - e35309.

5. Digital drip PCR (droplet digital PCR, ddPCR)

GNAQ and GNA11 mutations and downstream YAP activation in choroidal nevi / M.J.C. Vader, M.C. Madigan, M. Versluis, H.M. Suleiman, G. Gezgin, N.A. Gruis, J.J. Out-Luiting, W. Bergman, R.M. Verdijk, M.J. Jager, P.A. van der Velden // British Journal of Cancer. - 2017 .-- Vol. 117. - P. 884-887.

6. High Resolution Melting Curve Analysis

Mutation scanning of BRAF, NRAS, KIT, and GNAQ / GNA11 in oral mucosal melanoma: a study of 57 cases / J. Lyu, Y. Wu, C. Li, R. Wang, H. Song, G. Ren, W. Guo // J Oral Pathol Med. - 2016. - Vol. 45 (4). - P. 295-301.

Method 1 is widespread and quite informative. It provides complete information about the DNA sequence at the studied locus and allows one to determine de novo mutations, however, it does not have sufficient sensitivity when used for the analysis of somatic mutations (for this method, the sample should contain at least 25% mutant DNA), is characterized by low productivity and the need for expensive equipment and reagents. For further decoding of chromatograms, highly qualified personnel are required, which limits the use of this method.

Method 2 is one of the most advanced and high-performance approaches for the analysis of somatic mutations. It allows you to simultaneously obtain information about the nucleotide sequence of a large number of genes, to identify de novo mutations with high analytical sensitivity. Its disadvantages include the extremely high cost of reagents and equipment, the great complexity in preparing experiments and interpreting the results, which makes it unsuitable for routine laboratory use.

Method 3 is difficult in interpreting the results, provides only indirect information about the nucleotide sequence and is not subject to automation.

Method 4 gives an indirect idea of their nucleotide sequence of the locus. The analysis requires expensive equipment and highly qualified personnel, which reduces its convenience for routine use in clinical practice.

Method 5 has high analytical sensitivity, reliability and accuracy. Its disadvantage is the high cost of reagents and equipment.

Method 6 is very sensitive to the quality of the studied DNA samples; it provides indirect information about the nucleotide sequence of the studied locus; subsequent sequencing is required to accurately determine the mutation. It is more suitable for the study of terminal mutations and SNPs, rather than somatic mutations.

Thus, at the moment there is an urgent need to develop a method for diagnosing a significant number of mutations in the GNAQ and GNA11 genes, which would advantageously differ from known solutions by the simplicity of the analysis and low cost.

Currently, there are only a few patents for methods for analyzing mutations in the GNAQ and GNA11 genes.

WO 2016106391 A1 discloses systems, kits and methods for the quantitative detection of single nucleotide polymorphisms or mutations in the GNAQ and GNA11 genes for identifying malignant neoplasms. The method is based on the principle of real-time PNA-LNA-blocking PCR (PNA, peptide nucleic acid; LNA, locked nucleic acid). The methods include the use of PNA-based blocking probes that bind to the wild-type sequence of the target locus and prevent its amplification, and LNA-containing probes that are complementary to the mutant sequence of the target allele and fluoresce upon binding. This method requires parallel reactions to be performed for simultaneous analysis GNAQ and GNA11 genes.

Patent WO 2015116502 A1 describes an invention related to the use of metal nanoparticles for the early detection of uveal melanoma. Nanoparticles include a nucleic acid having a stem and loop structure that matches a metal domain on a fluorophore to quench a fluorophore. Upon binding to the sequence of the GNAQ variant, the structure of the stem and loop is destroyed and the fluorophore fluoresces. In specific embodiments, the metal domain contains gold. This method is not widely used in routine laboratory diagnostics.

Since all currently available methods for determining somatic mutations in the GNAQ and GNA11 genes have one or another disadvantage, there is a real need to create a simple, inexpensive, and specific method for detecting somatic mutations in these genes, with a view to further implementation in any standard clinical laboratory.

Such a method is provided by the present invention.

Disclosure of invention

The essence of the invention is to provide a method for determining somatic mutations in the GNAQ and GNA11 genes in benign and malignant neoplasms for diagnostic and prognostic purposes. The occurrence of somatic mutations in the GNAQ and GNA11 genes is an early event in the formation of uveal melanoma. Targeted drugs are currently being developed to treat patients with GNAQ / GNA11 mutations. The proposed invention may be applicable in clinical diagnostic laboratories of oncological institutions for diagnostic purposes and assess the prognosis of the development of the disease.

This method allows the detection of point somatic mutations Q209P (p. 626A> C), Q209L (p. 626A> T), Q209R (p. 626A> G) of the GNAQ gene and Q209L (p. 626A> T), Q209P (p. 626A> C) of the GNA11 gene.

The main features of this invention are LNA-blocking multiplex "nested" PCR and a biochip containing a set of immobilized differentiating oligonucleotides.

The first important feature of the invention are primers for amplification of the loci of the studied GNAQ and GNA11 genes, a set of which is used to obtain the studied fluorescently labeled DNA fragments in the required amount. The primer sequences are given in Sequence Listing 1 (SEQ ID NO: 1-6).

A second important feature of the invention are LNA oligonucleotides that recognize and specifically bind to wild-type DNA fragments, preventing their amplification. Due to this, DNA loci containing somatic mutations are predominantly amplified. The sequences of LNA oligonucleotides are shown in Sequence Listing 2 (SEQ ID NO: 7-8).

A third important feature of the invention is a biochip containing a set of immobilized differentiating oligonucleotides, the sequences of which are shown in Sequence Listing 3 (SEQ ID NO: 9-15). Differentiating oligonucleotides are immobilized in the cells of a hydrogel microchip, as described in the patent [Composition for immobilization of biological macromolecules in hydrogels, method for preparing the composition, biochip, method for PCR on a biochip / A.D. Mirzabekov, A.Yu. Rubina, S.V. Pankov, etc. // Patent RU 2206575 C2. - 2003] at a concentration of 200 μM. The layout of the biochip cells for the analysis of somatic mutations in the GNAQ and GNA11 genes is shown in FIG. one.

A set of primers and LNA oligonucleotides is used at the stage of preliminary amplification of DNA loci by conducting multiplex LNA-blocking “nested” PCR to prepare the target DNA for hybridization on a biochip. At the first stage, through the use of LNA-blocking multiplex PCR, amplification of DNA loci containing somatic mutations in the target sequences of the GNAQ and GNA11 genes takes place (if the sample contains mutations). In the second stage, single-stranded DNA is produced from the amplified fragment with the simultaneous introduction of a fluorescent label. Next, the hybridization of fluorescently labeled DNA fragments obtained after the second stage of PCR is carried out with oligonucleotides immobilized in gel cells located on a plastic substrate. After hybridization and washing of the biochip, an analysis of the obtained fluorescence pattern is carried out, based on which a conclusion is made about the genotype in the test sample. An example of a hybridization pattern is shown in FIG. 2.

Sequence Lists

List of sequences 1. Sequences of primers I and II stage for conducting multiplex PCR with subsequent analysis by hybridization on a biochip.

List of sequences 2. Nucleotide sequences of LNA oligonucleotides. Legend: capital letters - DNA nucleotides, lowercase letters - LNA nucleotides

List of sequences 3. Sequences of oligonucleotide probes used for immobilization in biochip cells.

List of figures

Figure 1. Scheme of a biochip for the analysis of somatic mutations in the GNAQ and GNA11 genes: Cells (M) containing the fluorescent dye Cy5, permanently glowing, are permanently illuminated for orientation and control of the luminescence intensity in the left-hand positions.

Figure 2. Hybridization pattern obtained using a biochip for the analysis of somatic mutations in the GNAQ and GNA11 genes in test No. XXX. The sample contains a Q209L mutation in the GNAQ gene.

The implementation of the invention

An object of the present invention is to provide an analysis method for detecting somatic mutations in the GNAQ and GNA11 genes.

To ensure the optimal composition and structure of the PCR primers used in the multiplex PCR protocol, primers are needed that do not form high-energy internal structures (studs and duplexes), provide specific amplification of the required amount of product, and are selected so that they are annealed on the target occurred at the same temperature. To ensure balanced effective amplification of the studied samples, parameters such as the concentration of MgCl ₂ and primers in the PCR mixture, the ratio of forward and reverse primers, the number of amplification cycles at both stages, the elongation, denaturation and annealing of primers at each stage should also be optimized. As a result of the work, primers of stages 1 and 2 were selected that allow efficient production of selected fragments of the GNAQ and GNA11 genes.

To ensure the optimal composition and structure of the LNA oligonucleotides used in the LNA-blocking multiplex PCR protocol, LNA oligonucleotides that do not form between themselves and with primers of high-energy internal structures (hairpins and duplexes), provide specific binding to the target sequence and when this is chosen so that under PCR conditions they form stable perfect duplexes with the target DNA and do not form imperfect duplexes. As a result of the work, LNA oligonucleotides were selected that allow the preferential production of selected fragments of the GNAQ and GNA11 genes carrying somatic mutations.

Oligonucleotides for immobilization on a biochip are selected in such a way as to identify all somatic mutations selected for analysis in the GNAQ and GNA11 genes.

Oligonucleotides must meet the following criteria: a discriminating probe must have high specificity for the mutant locus selected for analysis, which is a portion of a gene amplified by PCR with the fluorescent label turned on; the variable nucleotide should be in the middle region of the probe, since this position allows for greater discrimination between perfect and imperfect duplexes; the selected oligonucleotides should not contain highly stable secondary structures, the presence of which can lead to a decrease in the efficiency of hybridization.

On the biochip for determining somatic mutations in the GNAQ and GNA11 genes, 7 highly specific differentiating oligonucleotide probes (List of Sequences 3 (SEQ ID NO: 9-15)) are immobilized, the structure of which ensures binding only to fully complementary DNA targets, which causes a bright fluorescent signal corresponding biochip cells and gives the most clear picture of the distribution of hybridization signals. Nonspecific binding is minimized, which virtually eliminates false positive “triggering” of cells.

Since LNA-blocking PCR is used to amplify target DNA loci, cells containing probes with a "wild-type" sequence can have an extremely low level of fluorescence signal.

Here is the sequence of analysis using this method. The amplification of target sequences for subsequent hybridization on a biochip is carried out by means of a “nested” multiplex LNA-blocking PCR in two stages, and a DNA sample is used as a template for the reaction. Amplification of each DNA sample is carried out in one tube.

PCR can be carried out using any type of thermostable polymerase that does not have 5 ′> 3 ′ exonuclease activity (for example, SNPdetect polymerase (Eurogen)). To build a new chain, a mixture of dNTPs (dATP, dGTF, dTTP, dTTP) in the accepted concentrations is added to the buffer, and dUTP can be used instead of dTTP. For PCR, ready-made commercially available kits containing all the necessary components except primers of LNA oligonucleotides can be used.

At the first stage, LNA-blocking amplification of DNA loci of the GNAQ and GNA11 genes takes place with the predominant production of mutant fragments (if the sample contains a mutation). The product of the first PCR step is used as a matrix in the second step, which is carried out in a reaction mixture of the same composition, but LNA oligonucleotides are not added and an excess of reverse primers is added to obtain an excess of fluorescently labeled single-stranded PCR product capable of hybridization with allele-specific DNA probes on a biochip.

Primers, LNA oligonucleotides and oligonucleotide probes are synthesized using various chemical approaches, such as the phosphodiester method, hydrophosphoryl method, etc., while the most common is the phosphoamidite synthesis method. The synthesis of primers is carried out using automatic DNA / RNA synthesizers, for example, manufactured by Applied Biosystems (USA).

In the manufacture of the biochip, oligonucleotides that carry an active group providing immobilization at the 5'- or 3'-end can be used. Modification of the oligonucleotide for the introduction of the active group can be carried out both automatically in the synthesis using a wide range of commercially available modifiers, and postsynthetically in the manual mode. For example, in the synthesis of oligonucleotide probes using the 3'-Amino-Modifier C7 CPG 500 (Glen Research, USA), a free amino group spacer is introduced at the 3'-end of the oligonucleotides, which is used for subsequent immobilization of the oligonucleotide on a biochip.

For conducting multiplex LNA-blocking "nested" PCR, primers SEQ ID NO: 1-6 and LNA oligonucleotides SEQ ID NO: 7-8 are used, shown in the Lists of sequences 1 and 2.

The following primers and LNA oligonucleotides are used in the multiplex reaction of stage I: SEQ ID NO: 1, 2, 4, 5, 7, 8.

In the multiplex reaction of stage II, the following primers are used: SEQ ID NO: 1, 3, 4, 6.

Next, a hybridization of fluorescently labeled amplicons obtained after the second stage of PCR with immobilized oligonucleotides in the gel cells is carried out. Oligonucleotides are portions of the target sequences of the GNAQ and GNA11 genes and are complementary to the wild-type sequence or sequence containing the mutation.

Before staging the hybridization, the amplicon is denatured by heating the finished hybridization mixture at 95 ° C for 5 minutes. followed by rapid cooling on ice for 2 minutes. Hybridization can be carried out in any hybridization buffer known to those skilled in the art, for example, in a guanidine or SSPE buffer. A typical hybridization time is 12-14 hours at 37 ° C. The analysis of the genotype of the test sample is carried out taking into account the location of the oligonucleotide probes on the biochip, the scheme of which allows you to determine which somatic mutations are present in the sample.

The analyzed DNA fragment forms perfect hybridization duplexes only with oligonucleotides that are completely complementary to it. If the sequence of the analyzed DNA is completely complementary to the sequence of the probe, then a stable perfect duplex is formed (a fluorescence signal is detected). If the desired fragment is absent or there is a non-complementary base in it, then no stable duplex is formed (there is no fluorescence signal). Discrimination of perfect and imperfect duplexes is carried out after washing the biochip, comparing the intensities of the fluorescence signals of the corresponding cells of the biochip. Washing can be carried out in any buffer known in the art with the addition of salt (SSC, SSPE, etc.) or in deionized water, but in a shorter time [Molecular cloning: a laboratory manual / J.F. Sambrook, D.W. Russell // Cold Spring Harbor Laboratory Press. - 2001].

After washing the biochip, the results of hybridization are visualized using fluorescence excitation of the hybridized labeled PCR product. The hybridization pattern can be recorded using any detection system that recognizes a fluorescent signal (fluorescence microscope with a CCD camera, laser scanner, portable biochip analyzer, etc. commercially available fluorescence analyzers, for example, a portable biochip analyzer equipped with a CCD camera and special software manufactured by BIOCHIP-IMB LLC (Moscow, Russia).

Biochips can be made by sequentially applying a matrix of acrylamide gel cells to the surface of the glass substrate, activating the cells, and covalently immobilizing the modified oligonucleotides in the cells carrying active groups [Analysis of SNPs and other genomic variations using gel-based chips / A. Kolchinsky, A. Mirzabekov // Hum Mutat. - 2002. - Vol. 19. - P. 343-360. Review]. In addition to glass, another material can be used as a substrate, including metal, flexible membranes and plastic [The use of unmodified polymer materials for the manufacture of biochip substrates, a biochip based on them and a method for its manufacture, a method for immobilizing hydrogels on unmodified polymeric materials / C. B Pankov, E.Ya. Kreindlin, O.G. Somova, and others // Patent RU 2309959. - 2007]. Bochips can also be made by any other methods known to the person skilled in the art [Arrays of immobilized oligonucleotides - contributions to nucleic acids technology / H. Seliger, M. Hinz, E. Happ // Curr Pharm Biotechnol. - 2003. - Vol. 4. - P. 379-395].

For the manufacture of the biochip, the present invention uses the set of oligonucleotides SEQ ID NO: 9-15 shown in the sequence List 3. As a control of the passage of hybridization in the present invention, a control DNA sample is used. The location of specific oligonucleotide probes on the biochip can vary and is determined only by the convenience of interpreting the results of hybridization.

The following are examples that show the application of a method for analyzing somatic mutations in the GNAQ and GNA11 genes. It should be understood that the examples provided are for illustration only and are not intended to limit the scope of the claims expressed in the claims. Based on the present description, a person skilled in the art will be able to easily propose their own variants and modifications of the invention without departing from the general concept of the present invention and without involving their own inventive activity, so it should be understood that such variants and modifications will also be included in the scope of the claims of this inventions.

Example 1. Amplification of fragments of GNAQ and GNA11 genes by the method of "nested" LNA-blocking PCR in order to obtain a fluorescently labeled PCR product in the required amount.

From surgical material fixed in paraffin blocks, tumor tissue samples were obtained using manual microdissection under histological control. Genomic DNA was isolated using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany).

Locus of the GNAQ and GNA11 gene loci was carried out in a multiplex reaction, which contained the following primers and LNA oligonucleotides - SEQ ID NO: 1, 2, 4, 5, 7, 8. PCR was performed on a T100 device (Bio-Rad, USA) . The first-stage PCR mixture with a total volume of 25 μl included: 1 × PCR buffer (67 mM Tris-HCl, pH 8.6, 166 mM (NH ₄ ) ₂ SO ₄ , 0.01% Triton X-100), 1.5 mM MgCl ₂ , 0.2 mM each of dNTPs (Sileks, Russia), 5 U SNPdetect polymerase (Eurogen, Russia), 0.2 μM primers, 0.04 μM LNA oligonucleotides and 25 ng DNA. Amplification was carried out according to the following scheme: denaturation at 94 ° C (3 min 30 s), then 35 cycles: 94 ° C (30 s), 62 ° C (20 s), 72 ° C (10 s), then elongation at 72 ° C for 3 minutes The mixture of the second stage of PCR differed in the composition and concentration of primers. It contained 0.2 μM forward primers (SEQ ID NO: 1, 4), and 2 μM reverse primers (SEQ ID NO: 3, 6) and did not contain LNA oligonucleotides. For fluorescence labeling of the second round PCR product, the mixture contained 0.2 nM fluorescently labeled dUTP-Su5, which was integrated into the chain during amplification. As a matrix, 2 μl of the PCR product of stage I was added to the mixture and amplification was carried out according to the following scheme: denaturation at 94 ° С (3 min 30 s), then 35 cycles: 94 ° С (30 s), 62 ° С (20 s) , 72 ° С (10 s), then elongation at 72 ° С for 3 min.

Example 2. Oligonucleotide biochip for determining somatic mutations in the GNAQ and GNA11 genes.

Microchip immobilizing oligonucleotides are synthesized on a 394 DNA / RNA Synthesizer (Applied Biosystems, USA) using a standard phosphoamidite procedure. The 5 ′ or 3 ′ end of the oligonucleotides contains a free amino group spacer, which is introduced into the oligonucleotide by synthesis using the 3′-Amino-Modifier C7 CPG 500 (Glen Research), USA). The addition of a fluorescent label to the free amino group oligonucleotides is carried out in accordance with the manufacturer's recommendations. Oligonucleotides labeled with cyanine dye Su-3 are purified by HPLC from oligonucleotides that do not include a fluorescent dye on a Hypersil ODS 5 μm, 4.6 × 250 mm column (Sypelco Int, USA).

The biochip is made by copolymerization of an oligonucleotide in an acrylamide gel, as described previously [The use of unmodified polymer materials for the manufacture of biochip substrates, a biochip based on them and a method for its manufacture, a method for immobilizing hydrogels on unmodified polymeric materials / C.V. Pan'kov, E.Ya. Kreindlin, O.G. Somova et al. // Patent RU 2309959. - 2007.], [Method for the immobilization of oligonucleotides containing unsaturated groups in polymer hydrogels during microchip formation / A.D. Mirzabekov, A.Yu. Rubina, S.V. Pankov, etc. // Patent RU 2175972. - 2001.]. After manufacturing, biochips undergo application quality control, which includes visual control of the physical sizes of gel cells using a light microscope in reflected light and monitoring the concentration of immobilized oligonucleotides using Su-3 dye (BIOCHIP-IMB LLC, Moscow, Russia). The biochip contains 7 immobilized oligonucleotide probes (SEQ ID NO: 9-15), a list of which is also presented in the List of sequences 3. The cells are applied according to the scheme in FIG. 1. Example 3. Hybridization of a labeled product on a biochip

The reaction mixture obtained after stage II PCR described in Example 1 is used for hybridization on a biochip. The hybridization mixture with a total volume of 40 μl contained 10 μl of formamide (Serva, USA), 10 μl of 20 × SSPE (Promega, USA) and 20 μl of the amplification. The prepared hybridization mixture was denatured at 95 ° C for 5 minutes before hybridization, and cooled in ice for 2 minutes. and applied to the biochip in a hybridization chamber with a volume of 40 μl (BIOCHIP-IMB LLC, Moscow, Russia). Hybridization is carried out for 12-14 hours at a temperature of 37 ° C. After incubation is complete, the hybridization chamber is removed along with the unreacted hybridization mixture and washed in 1 × SSPE buffer (Promega, USA) at room temperature for 15 minutes.

Example 4. Registration and interpretation of hybridization results

The hybridization pattern is recorded using a portable biochip analyzer equipped with a CCD camera manufactured by Biochip-IMB LLC. A description of an automatic image analysis algorithm using Image Ware ™ is beyond the scope of the present invention.

We determine the genotype from the hybridization pattern shown in FIG. 2.

In the biochip cells corresponding to the 209 codon of the GNAQ gene, an intense fluorescent signal is observed only in a pair of cells corresponding to the somatic mutation Q209L. Biochip cells corresponding to the “wild type” of a given locus have a low level of fluorescence. For the GNA11 gene, a fluorescent signal is observed only in a pair of cells corresponding to the sequence of the "wild type". Therefore, the analyzed sample contains the Q209L mutation in the GNAQ gene.

Claims (10)

1. Method for detecting somatic mutations Q209P (p. 626A> C), Q209L (p. 626A> T), Q209R (p. 626A> G) in the GNAQ gene and Q209L (p. 626A> T), Q209P (p. 626A > C) in the GNA11 gene, comprising the following stages: (a) amplification of fragments of the GNAQ and GNA11 genes using LNA-blocking multiplex "nested" PCR, in which a sample of tumor DNA is used as an amplification matrix: in the first step of PCR, primers of SEQ ID NO: 1, 2, 4, 5 and LNA oligonucleotides of SEQ ID NO: 7, 8 are used; at the second stage of PCR, primers are used SEQ ID NO: 1, 3, 4, 6; as a result, a predominantly single-chain fluorescently labeled PCR product is formed; (b) providing a biochip for identifying somatic mutations in the GNAQ and GNA11 genes containing a set of immobilized oligonucleotides with sequences of SEQ ID NO: 9-15; (c) hybridization of the labeled PCR product on a biochip; (d) recording and interpretation of hybridization results. 2. The method according to p. 1, in which the registration of the results of hybridization is carried out using a device capable of recording and recording fluorescence signals, equipped with software that allows automatic processing of signal intensity with subsequent interpretation of the results. 3. The method according to p. 1, in which to identify somatic mutations in the GNAQ and GNA11 genes, amplified using a set of primers and LNA oligonucleotides, characterized in p. 1, a biochip is used, which is a substrate with a set of oligonucleotides immobilized on it with sequences SEQ ID NO: 9-15.

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