RU2552483C1 - Method of analysis of somatic mutations in genes egfr, kras and braf using lna-blocking multiplex pcr and subsequent hybridisation with oligonucleotide biological microchip (biochip) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method comprises amplification of fragments of genes EGFR, KRAS and BRAF using LNA-blocking multiplex "nested" PCR in which the template for amplification is used as DNA tumour sample. The single-stranded fluorescently labelled PCR product is obtained. The biochip for identification of somatic mutations in the genes EGFR, KRAS and BRAF is obtained. Hybridisation of fluorescently labelled PCR-product in the biochip is carried out. The results of hybridisation on the biochip are recorded and interpreted.

EFFECT: invention enables to carry out analysis in a short period of time and with low material costs.

4 cl, 2 dwg, 4 tbl, 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 the sensitivity of a number of oncological diseases to targeted therapy by analyzing somatic mutations in the EGFR, KRAS and BRAF genes using LNA-blocking multiplex PCR technology and subsequent hybridization with an oligonucleotide biological microchip (biochip) .

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 can be distinguished:

1) Enzymatic approaches:

amplified fragment length polymorphism (AFLP);

restriction fragment length polymorphism (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 sequencing

high throughput sequencing (NGS).

2) Chemical methods:

chemical splitting of heteroduplexes;

chemical ligation.

3) 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.

4) Solid phase detection:

hybridization on oligonucleotide matrices;

fiber optic DNA hybridization analysis;

elongation of immobilized primers (minisequencing);

pyrosequencing.

5) Chromatographic methods:

high performance liquid chromatography (HPLC).

6) Physical methods:

mass spectrometry;

resonance fluorescence quenching (FRET);

luminescence, depending on the local environment.

The most common methods presented are:

1. Sequencing of amplified DNA fragments according to Senger.

Frequent epidermal growth factor receptor gene mutations in malignant pleural effusion of lung adenocarcinoma / S-G. Wu, C-H. Gow, C-J. Yu et al. // Eur Respir J. - 2008 .-- Vol. 32. - P. 924-930.

Detection of Epidermal Growth Factor Receptor Mutation in Transbronchial Needle Aspirates of Non-Small Cell Lung Cancer / A. Horiike, H. Kimura, K. Nishio et al. // Chest. - 2007. - Vol. 131. - P. 1628-1634.

Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib / T.J. Lynch, D.W. Bell, R. Sordella et al. // N Engl J Med. - 2004. - Vol. 350. - P. 2129-2139.

A comparison of Direct sequencing, Pyrosequencing, High resolution melting analysis, TheraScreen D × S, and the K-strip StripAssay for detecting KRAS mutations in non small cell lung carcinomas / S. Jancik, J. Drabek, J. Berkovcova et al. // Journal of Experimental & Clinical Cancer Research - 2012 .-- Vol. 31 .-- 79.

Sequencing analysis of BRAF mutations in human cancers / R. Wooster, A.P. Futreal, M.R. Stratton // Methods Enzymol. - 2006. - Vol. 407. - P. 218-224.

2. Conformational analysis of single-stranded DNA fragments (SSCP).

EGFR gene amplification is related to adverse clinical outcomes in cervical squamous cell carcinoma, making the EGFR pathway a novel therapeutic target / K. Iida, K. Nakayama, M.T. Rahman et al. // Br J Cancer. - 2011 .-- Vol. 105. - P. 420-427.

EGFR Mutations in Non-Small-Cell Lung Cancer: Analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment / A. Marchetti, C. Martella, L. Felicioni et al. // J Clin Oncol. - 2005. - Vol. 23. - 857-865.

High Prevalence of BRAF Mutations in Thyroid Cancer: Genetic Evidence for Constitutive Activation of the RET / PTC-RAS-BRAF Signaling Pathway in Papillary Thyroid Carcinoma / E.T. Kimura, M.N. Nikiforova, Z. Zhu et al. // Cancer Res. - 2003. - Vol. 63. - P. 1454-1457.

3. Allele-specific PCR (AS-PCR).

Two allele-specific PCR assays for screening epidermal growth factor receptor gene hotspot mutations in lung adenocarcinoma / R. Dahse, A. Berndt, A.K. Dahse et al. // Mol Med Report. - 2008. - Vol. 1. - P. 45-50.

PCR-based testing for therapy-related EGFR mutations in patients with non-small cell lung cancer / R. Dahse, A. Berndt, H. Kosmehl // Anticancer Res. - 2008. - Vol. 28 .-- 2265-2270.

KRAS mutations are associated with inferior clinical outcome in patients with metastatic colorectal cancer, but are not predictive for benefit with cediranib / J.C. Smith, L. Brooks, P.M. Hoff et al. // Eur J Cancer. - 2013 .-- Vol. 49. - P. 2424-2432.

4. Hybridization with biochips.

Combined detection of p53, p16, Rb, and EGFR mutations in lung cancer by suspension microarray / Y. Ye, D. Wang, C. Su et al. // Genet Mol Res. - 2009. - Vol. 8. - P. 1509-1518.

The KRAS StripAssay for detection of KRAS mutation in Egyptian patients with colorectal cancer (CRC): a pilot study / Y. Abd El Kader, G. Emera, E. Safwat et al. // J Egypt Natl Canc Inst. - 2013 .-- Vol. 25. - P. 37-41.

5. SMart amplification.

Correlation between computed tomography findings and epidermal growth factor receptor and KRAS gene mutations in patients with pulmonary adenocarcinoma / M. Sugano, K. Shimizu, T. Nakano et al. // Oncol Rep. - 2011 .-- Vol. 26. - P. 1205-1211.

Rapid Detection of Epidermal Growth Factor Receptor Mutations in Lung Cancer by the SMart-Amplification Process / K. Hoshi, H. Takakura, Y. Mitani et al. // Clin Cancer Res. - 2007. - Vol. 13. - P. 4974-4983.

6. PNA-blocking real-time PCR.

PNA-mediated Real-Time PCR Clamping for Detection of EGFR Mutations / J. Choi, M. Cho, M. Oh et al. // Bull. Korean Chem. Soc. - 2010 .-- Vol. 31. - P. 3525-3529.

7. LNA-blocking real-time PCR.

Rebiopsy of Lung Cancer Patients with Acquired Resistance to EGFR Inhibitors and Enhanced Detection of the T790M Mutation Using a Locked Nucleic Acid-Based Assay / G.J. Riely, S.B. Solomon, M.F. Zakowski et al. // Clin Cancer Res. - 2011 .-- Vol. 17. - P. 1169-1180.

8. PNA-LNA-blocking PCR in real time.

Reliable detection of rare mutations in EGFR gene codon L858 by PNA-LNA PCR clamp in non-small cell lung cancer / M. Skronski, J. Chorostowska-Wynimko, E. Szczepulska et al. // Adv Exp Med Biol. - 2013 .-- Vol. 756. - P. 321-331.

First-Line Gefitinib for Patients With Advanced Non-Small-Cell Lung Cancer Harboring Epidermal Growth Factor Receptor Mutations Without Indication for Chemotherapy / A. Inoue, K. Kobayashi, K. Usui et al. // J Clin Oncol. - 2009. - Vol. 27. - P. 1394-1400.

Peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp-based detection test for gefitinib-refractory T790M epidermal growth factor receptor mutation / H. Miyazawa, T. Tanaka, Y. Nagai et al. // Cancer Sci. - 2008. - Vol. 99. - P. 595-600.

Genetic Heterogeneity of the Epidermal Growth Factor Receptor in Non-Small Cell Lung Cancer Cell Lines Revealed by a Rapid and Sensitive Detection System, the Peptide Nucleic Acid-Locked Nucleic Acid PCR Clamp // Y. Nagai, H. Miyazawa, Huqun et al. // Cancer Res. - 2005. - Vol. 65. - P. 7276-7282.

9. Capillary electrophoresis.

EGFR mutations in lung adenocarcinomas: clinical testing experience and relationship to EGFR gene copy number and immunohistochemical expression / A.R. Li, D. Chitale, G.J. Riely et al. // J Mol Diagn. - 2008. - Vol. 10 .-- P. 242-248.

10. PCR enriched in mutant sequence.

Detection of EGFR gene mutation in lung cancer by mutant-enriched polymerase chain reaction assay / H. Asano, S. Toyooka, M. Tokumo et al. // Clin Cancer Res. - 2006. - Vol. 12. - P. 43-48.

11. Analysis of melting curves of high resolution.

High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies / H. Do, M. Krypuy, P.L. Mitchell et al. // BMC Cancer. - 2008. - Vol. 8.142.

High-Resolution Melting Analysis for Rapid Detection of KRAS, BRAF, and PIK3CA Gene Mutations in Colorectal Cancer / L. Simi, N. Pratesi, M. Vignoli et al. // Am J Clin Pathol. - 2008. - Vol. 130. - P. 247-253.

Method 1 is widespread and quite informative. It provides complete information about the DNA sequence at the studied locus and allows the determination of de novo mutations. However, it does not have sufficient sensitivity for use in clinical practice in the analysis of somatic mutations (for this method, the sample must contain at least 25% mutant DNA), is characterized by low productivity and the need for expensive equipment and reagents. In addition, for further decoding of chromatograms, highly qualified personnel are required, which prevents the use of this method in large-scale studies.

Method 2 is complicated in the execution and interpretation of the results, gives only indirect information about the nucleotide sequence and is not subject to automation.

Method 3 is widespread enough, but it requires the formulation of independent parallel PCR reactions corresponding to the number of analyzed single nucleotide substitutions. To evaluate the results, gel electrophoresis is also required, which dramatically reduces its value for large-scale genotyping.

Method 4 is the most promising for simultaneous genotyping of a large number of genetic loci, however, at the moment there are no cheap test systems based on this method that would allow for most analyzes to determine the majority of clinically important mutations in the studied gene.

Method 5 is quick to execute, but requires parallel reactions in terms of the number of mutations analyzed. The disadvantages of this method include the fact that with its help only known mutations can be detected.

Methods 6-8 also require setting up several independent responses for each patient. These methods allow only the fact of the presence / absence of a mutation in the sample to be detected, but does not provide information about the nucleotide sequence of the analyzed loci. If the presence of only one mutation is analyzed in one tube, this approach is justified, however, if several types of mutations are analyzed simultaneously in one tube (for example, several different deletion variants in exon 19 of EGFR), this method is not sufficiently informative.

Method 9 makes it possible to identify deletions with high accuracy and reliability, however, it gives only an indirect idea of their nucleotide structure. When determining single nucleotide substitutions, additional manipulations are required (in particular, the use of restriction enzymes), which significantly increases the analysis time and reduces its convenience for routine use in clinical practice.

Method 10 has a fairly high sensitivity, but requires a large number of manipulations. In addition, it also requires the setting of several parallel reactions for each patient.

Method 11 allows you to diagnose mutations in the EGFR gene with high accuracy and reliability, however, equipment for it is quite expensive.

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

Currently, there are many patents for methods for analyzing mutations in the EGFR, KRAS, BRAF genes associated with tumor sensitivity to targeted therapy. Typically, such methods are focused on determining the presence / absence of mutations in the test sample and do not determine the individual structure of the genetic change (for example, Chinese patent CN 101565742). In the case of point mutation genotyping (for example, L858R and T790M EGFR), this approach may well be justified, however, when analyzing deletions in exon 19 of EGFR, which do not have a conservative structure, the researcher does not receive information about which deletion is present in the clinical sample. Recent studies have shown that different deletions in exon 19 of the EGFR gene have different effects on the effectiveness of antitumor therapy.

Many patents describing methods for analyzing mutations in the EGFR gene include only markers associated with increased sensitivity of the tumor to therapy, as, for example, in patent applications WO 2011035538 and WO 2009129693, but do not include resistance markers.

In Chinese patent CN 101041850 and American patent application WO 2006086777, on the contrary, a method for analyzing only the T790M EGFR mutation, which is a marker of tumor resistance to anti-EGFR therapy, is proposed. Patent application WO2006086777 describes a method which consists in amplifying the nucleotide sequence of an EGFR gene including the 790 codon, processing the amplification products with the restriction enzyme MaHI, which selectively cleaves only the sequence carrying the single nucleotide substitution in the codon, and subsequent detection of the restriction products using a capillary electrophore ABI 3100 Avant, Applied Biosystems, Foster City, California, United States). Analysis of this mutation alone is not sufficient to select targeted antitumor therapy for non-small cell lung cancer. But this analysis may be useful in monitoring the patient’s treatment with anti-EGFR drugs, since this somatic mutation often occurs in the tumor during treatment.

CN 1687457 describes a method for determining mutations in 18-21 exons of the EGFR gene, including amplification using HotStarTaq Master Mix and subsequent sequencing in two directions. It is a modification of method 1. To use this method requires expensive equipment and highly qualified personnel, which makes it unsuitable for use in routine clinical diagnostics.

Japanese patent JP 2007202546 describes primers that selectively amplify only the mutant sequence of the EGFR gene. To visualize amplification products, it is proposed to use agarose gel electrophoresis (method 3).

GB 2424886 provides a set of primers for amplifying mutations or polymorphisms in the EGFR gene. These primers can be used to analyze the EGFR gene using the ARMS technique (amplification-refractory mutation system, amplification of the refractory mutation system). This method has found rather wide application for the detection of mutations in a number of hereditary diseases, however, difficulties in selecting primers and in choosing the optimal PCR regime limit its widespread use.

Many patents offer various options for setting up real-time PCR. In US patent US 2013095491 and in European EP 2540838 presents a method for determining mutations using allele-specific PCR in the BRAF gene. Allele-specific PCR based methods for determining mutations in the EGFR gene are described in Chinese patents CN 102021228 and CN 101565742 (genotyping is performed in 19 and 21 exons). In Chinese patents CN 101434985, CN 101899496, CN 102041313, reagent kits based on the real-time PNA blocking PCR method for detecting mutations in various KRAS codons are presented. Such a large number of test systems based on real-time PCR is explained by the fact that this method is quite easy to learn, often does not require additional skills and expensive reagents, but it requires the purchase of expensive equipment. Also, its use in clinical practice with the simultaneous analysis of many mutations is a rather time-consuming process.

In Chinese patents CN 102443140, CN 101070538 and CN 101092644 a method for amplification of fragments of the EGFR gene is proposed.

Russian patent RU 2454464 provides a method for genotyping mutations in the EGFR gene using allele-specific PCR with visualization of amplification products using polyacrylamide gel electrophoresis (method 3). This method has high sensitivity, specificity, does not require specific equipment, expensive reagents and highly qualified personnel. But the formulation of polyacrylamide gel electrophoresis significantly reduces the potential for clinical use of this approach.

Using various amplification methods, mutations in other genes are also analyzed. In the patent application WO2005071109, mutations in the MSH, MLH genes and the T1796A mutation (V600E) in the BRAF gene are analyzed using a combination of SSCP and electrophoresis. Patent Application WO 2012068562 describes a method for genotyping mutations in the BRAF gene in melanoma patients by sequencing. In patent application WO2005066346, mutations are detected using the SSCP method at the 599 codon of the BRAF gene and at 12, 13 codons of the KRAS gene. This method of genotyping has all of the above advantages and disadvantages.

Several patents and patent applications have been published that show various technologies for determining mutations by hybridization with biochips (method 4). Korean patent KR 101133 242 describes a method for hybridizing EGFR gene fragments with probes located on gold, silver or platinum nanoparticles. This method has high accuracy, but also high cost. US Pat. No. 7,833,757 discloses a method for analyzing mutations in the APC, KRAS, beta-catenin, BRAF genes in colon cancer by hybridization with oligonucleotides immobilized on a microchip and subsequent detection using the biotin-streptavidin system. WO2009129693 describes a method for analyzing 19 and 21 exons of the EGFR gene using liquid biochip technology. The analysis has high accuracy and high speed. In Chinese patent CN 102443626, the most complete genotyping technique for lung cancer is reflected. Using a combination of fluorescence PCR technology and hybridization with probes, mutations in the EGFR, KRAS, BRAF genes, as well as EML4-ALK translocation, are analyzed. The analysis has good sensitivity, specificity and high speed.

Since all currently available methods for determining somatic mutations in the EGFR, KRAS, and BRAF genes have some drawbacks, there is a real need to create a simple, inexpensive, and specific method for detecting somatic mutations associated with tumor sensitivity to targeted therapy, 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 genes EGFR, KRAS and BRAF, affecting the sensitivity of the tumor to targeted therapy. This method allows the detection of somatic point mutations G12S, G12A, G12C, G12D, G12R, G12V, G13D, G13V, G13A, G13C, G13R, Q61L, Q61H of the KRAS gene, V600E of the BRAF gene, G719A, G719C, G719S, T785, G719S, T7 EGFR and deletions in exon 19 EGFR: E746-A750del (2 options: c. 2235-2249del, c. 2236-2250del), E746-T751del ins A, E746-T751del ins V, E746-S752del ins V, L747-A750del ins P, L747-T751del ins P, L747-T751del, L747-P753del ins S, E746-S752del ins V, E746-A750del insQP, E746-T751del ins VA, E746-P753del ins VS.

The potential effects of somatic mutations in the EGFR, KRAS and BRAF genes on the effectiveness of treatment with targeted antitumor drugs are presented in table. one.

References to Table 1

1. Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib / S.W. Han, T.Y. Kim, P.G. Hwang, et al. // J Clin Oncol. - 2005. - Vol. 23. - P. 2493-2501.

2. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib / T.J. Lynch, D.W. Bell, R. Sordella et al. // N Engl J Med. - 2004. - Vol. 350. - P. 2129-2139.

3. Mutations in the tyrosine kinase domain of the EGFR gene associated with gefitinib response in non-small-cell lung cancer / R. Rosell, Y. Ichinose, M. Taron et al. // Lung Cancer. - 2005. - Vol. 50. - P. 25-33.

4. Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib-treated chemorefractory lung adenocarcinomas / M. Taron, Y. Ichinose, R. Rosell et al. // Clin Cancer Res. - 2005. - Vol. 11. - P. 5878-5885.

5. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib / S. Kobayashi, T.J. Boggon, T. Dayaram et al. - New Eng. J Med. - 2005. - Vol. - 352. - P. 786-792.

6. Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR / D.W. Bell, I. Gore, R.A. Okimoto et al. // Nat Genet. - 2005. - Vol. 37. - P. 1315-1316.

7. EGFR mutation and response of lung cancer to gefitinib / S. Toyooka, K. Kiura, T. Mitsudomi // N Engl J Med. - 2005. - Vol. 352. - author reply 2136.

8. Response to erlotinib and prognosis for patients with de novo epidermal growth factor receptor (EGFR) T790M mutations / G.J. Riely, H.A. Yu, M.E. Arcila et al. // J Clin Oncol. - 2013 .-- Vol. 31. - Abstr 8018.

9. Biomarker analyses and final overall survival results from a phase III, randomized, open-label, first-line study of gefitinib versus carboplatin / paclitaxel in clinically selected patients with advanced non-small-cell lung cancer in Asia (IPASS) / M Fukuoka, YL Wu, S. Thongprasert et al. // J Clin Oncol. - 2011 .-- Vol. 29. - P. 2866-2874.

10. Clinicopathologic Significance of the Mutations of the Epidermal Growth Factor Receptor Gene in Patients with Non-Small Cell Lung Cancer / Y. Tomizawa, H. Iijima, N. Sunaga et al. // Clin Cancer Res. - 2005 .-- vol. 11. - P. 6816-6822.

11. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy / J.G. Paez, P.A. Janne, J.C. Lee et al. // Science. - 2004. - Vol. 304. - P. 1497-1500.

12. EGF receptor gene mutations are common in lung cancers from ′ never smokers ′ and correlate with sensitivity of tumors to gefitinib (Iressa) and erlotinib (Tarceva) / W. Pao, V. Miller, M.F. Zakowski // Proc Natl Acad Sci USA. - 2004. - Vol. 101. - P. 13306-13311.

13. Biological and clinical significance of KRAS mutations in lung cancer: an oncogenic driver that contrasts with EGFR mutation / K. Suda, K. Tomizawa, T. Mitsudomi // Cancer Metastasis Rev. - 2010 .-- Vol. 29. - P. 49-60.

14. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer / A. Lièvre, J.B. Bachet, D. Le Corre et al. // Cancer Res. - 2006. - Vol. 66. - P. 3992-3995.

15. A patient with BRAF V600E lung adenocarcinoma responding to vemurafenib / O. Gautschi, C. Pauli, K Strobel et al. // J Thorac Oncol. - 2012. - Vol. 7 (10): e23-24.

16. Molecular characterization of acquired resistance to the BRAF inhibitor dabrafenib in a patient with BRAF-mutant non-small-cell lung cancer / C.M. Rudin, K. Hong, M. Streit et al. // J Thorac Oncol. - 2013 .-- Vol. 8.- e41-42. doi: 10.1097 / JTO.0b013e31828bb1b3.

17. Analysis of PTEN, BRAF, and EGFR status in determining benefit from cetuximab therapy in wild-type KRAS metastatic colon cancer / P. Laurent-Puig, A. Cayre, G. Manceau et al. // J Clin Oncol. - 2009. - Vol. 27. - P. 5924-5930.

18. Clinical development of dabrafenib in BRAF mutant melanoma and other malignancies / G.T. Gibney, J.S. Zager // Expert Opinion on Drug Metabolism & Toxicology. - 2013 .-- Vol. 9 (7). - P. 893-899. doi: 10.1517 / 17425255.2013.794220.

19. B-Raf and the inhibitors: From bench to bedside / T. Huang, M. Karsy, J. Zhuge et al. // Journal of Hematology & Oncology. - 2013 .-- Vol. 6. - P. 30. doi: 10.1186 / 1756-8722-6-30.

20. Improved survival with vemurafenib in melanoma with BRAF V600E mutation / P.B. Chapman, A. Hauschild, C. Robert et al. // N Engl J Med. - 2011 .-- Vol. 364. - P. 2507-2516. doi: 10.1056 / NEJMoa1103782.

21. http://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm354478.htm

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

The first important aspect of the invention are primers for amplification of DNA loci of the studied genes EGFR, KRAS, BRAF, a set of which is used to obtain the studied fluorescently labeled DNA fragments in the required amount. The sequence of the primers are given in table. 2 and the List of sequences (SEQ ID NO: 1-25).

A second important aspect of the invention are LNA oligonucleotides that provide preferential amplification of DNA loci containing somatic mutations. The predominant amplification of mutant DNA loci occurs as a result of the fact that LNA oligonucleotides recognize and specifically bind to wild-type DNA fragments, preventing their amplification. The sequence of LNA oligonucleotides are given in table. 3 and the List of sequences (SEQ ID NO: 26-32).

A third important aspect of the invention is a biochip containing a set of immobilized differentiating oligonucleotides, the sequences of which are given in table. 4 and the List of sequences (SEQ ID NO: 33-72). 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 et al. // Patent RU 2206575 C2. - 2003] at a concentration of 200-400 μm. The arrangement of biochip cells for the analysis of somatic mutations in the EGFR, KRAS, and BRAF gene 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 EGFR, KRAS, and BRAF 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, a 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.

The implementation of the invention

An object of the present invention is to provide a rapid analysis method for detecting somatic mutations in the EGFR, KRAS, and BRAF genes.

To ensure the optimal composition and structure of the PCR primers used in the multiplex PCR protocol, primers are necessary 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 for all. To ensure balanced effective amplification of the studied samples, parameters such as the concentration of MgCl ₂ and PCR 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 the 1st and 2nd stages were selected that allow the efficient production of selected fragments of the EGFR, KRAS, and BRAF 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 EGFR, KRAS, and BRAF genes carrying somatic mutations.

Immobilization oligonucleotides on a biochip to predict the effectiveness of treatment of patients with targeted drugs are selected in such a way as to identify all somatic mutations selected for analysis in the EGFR, KRAS and BRAF genes.

Oligonucleotides must meet the following criteria:

1) The discriminating probe should have high specificity for the mutant locus selected for analysis, which is a portion of a gene amplified by PCR with a fluorescent label on.

2) The variable nucleotide should be in the middle region of the probe, since this position allows for greater discrimination between perfect and imperfect duplexes.

3) The selected oligonucleotides should not contain highly stable secondary structures, the presence of which can lead to a decrease in the efficiency of hybridization.

40 highly specific differentiating oligonucleotide probes (Table 4 and the List of sequences (SEQ ID NO: 33-72)) are immobilized on a biochip for determining somatic mutations in the EGFR, KRAS, and BRAF genes, the structure of which ensures binding only to fully complementary DNA targets, which causes a bright fluorescent signal in the corresponding cells of the biochip 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 the DNA loci of interest to us, 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 PCR products 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 two tubes: in the first tube, the fragments of the EGFR gene are amplified, and in the second, the fragments of the KRAS and BRAF genes.

PCR can be carried out using any type of thermostable polymerase (Taq polymerase, Tth polymerase, Tfl polymerase, Pfu polymerase, Vent polymerase, DeepVent polymerase, etc. commercially available enzymes isolated from thermophiles) operating in appropriate buffer. 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 can be used.

At the first stage, LNA-blocking amplification of DNA loci of the EGFR, KRAS, and BRAF genes takes place with the predominant production of mutant fragments (if the sample contains a mutation). The product of the first stage of PCR is used as a matrix in the second stage, which is carried out in a reaction mixture of the same composition, but LNA oligonucleotides are not added and an excess of one of the primers is added to obtain an excess of a fluorescently labeled single chain PCR product capable of hybridization with allele-specific DNA probes on a biochip. As a fluorescent label, deoxynucleotide triphosphate is used, namely Su5-dUTP, which is inserted into the de novo synthesized DNA strand. Any fluorochrome without restriction (for example, FITC, Texas red, Su-3, etc.), as well as biotin, can also be used as a fluorescent label.

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 can be used that carry an active group at the 5 ′ or 3 ′ end that provides immobilization. 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′-Ammo-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.

To conduct multiplex LNA-blocking "nested" PCR, primers SEQ ID NO: 1-25 and LNA oligonucleotides SEQ ID NO: 26-32 are used, are given in the List of sequences in table. 2 and 3.

The following primers and LNA oligonucleotides are used in the multiplex reactions of stage I: the first multiplex reaction (SEQ ID NO: 1, 2, 5, 6, 9, 10, 13, 14, 26-29), the second multiplex reaction (SEQ ID NO: 17, 18, 20, 21, 23, 24, 30-32).

The following primers are used in the multiplex reaction of stage II: the first multiplex reaction (SEQ ID NO: 3, 4, 7, 8, 11, 12, 15, 16), the second multiplex reaction (SEQ ID NO: 17, 19, 20, 22, 23, 25).

Next, hybridization of the fluorescently labeled DNA fragments obtained after the second PCR step is carried out with oligonucleotides immobilized in the gel cells, which are portions of the target sequences of the EGFR, KRAS and BRAF genes, and are complementary to the wild-type sequence or the sequence containing mutations.

Before setting 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. Typical hybridization times are 12-14 hours at 37 ° C. The analysis of genotypes and alleles in the test sample is carried out taking into account the location of oligonucleotide probes on the biochip, the scheme of which allows you to determine which somatic mutations are present in a particular 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 an incomplete base in it, 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 hybridization and washing of the biochip, the results of hybridization are visualized by excitation of fluorescence of the hybridized labeled PCR product of the second round of PCR, based on which a conclusion is made about the genotype in the test sample. 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 a 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.V. . 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 in the present invention uses a set of oligonucleotides SEQ ID NO: 33-72 shown in the List of sequences, as well as in table. 4. As a control of passage of hybridization in the present invention use a sample of control DNA. 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 use of a method for analyzing somatic mutations in the EGFR, KRAS, and BRAF gene associated with the sensitivity of lung cancer to antitumor targeted therapy. 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 the EGFR, KRAS and BRAF genes by the method of "nested" LNA-blocking PCR in order to obtain the 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).

The generation of EGFR, KRAS, and BRAF gene loci was carried out in two parallel multiplex reactions. The first multiplex reaction contained the following primers and LNA oligonucleotides — SEQ ID NO: 1, 2, 5, 6, 9, 10, 13, 14, 26-29; the second multiplex reaction is SEQ ID NO: 17, 18, 20, 21, 23, 24, 30-32. PCR was performed on a Dyad instrument (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), 2.5 U Taq polymerase (Sileks, Russia), 0.2 μM primers, 0.05 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 (first multiplex: SEQ ID NO: 3, 7, 11, 12, 15; second multiplex: SEQ ID NO: 17, 20, 23), and 2 μM reverse primers (first multiplex: SEQ ID NO: 4, 8, 12, 16; the second multiplex: SEQ ID NO: 19, 22, 25) and did not contain an LNA oligonucleotide. 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 performed 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 min.

Example 2. Oligonucleotide biochip for determining somatic mutations in the EGFR, KRAS and BRAF 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 column 5 μm, 4.6 × 250 mm (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]. Pankov, E.Ya. Kreindlin, O.G. Somova, etc. // 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 et al. // 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 40 immobilized oligonucleotide probes (SEQ ID NO: 33-72), a list of which is also presented in table. 4. The cells are applied according to the circuit of FIG. one.

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 10 μl of the amplification from the first multiplex reaction and 10 μl of the amplification from the second multiplex reaction. Before hybridization, the prepared hybridization mixture was denatured at 95 ° C for 5 min, cooled in ice for 2 min, and applied onto a biochip in a 40 μl hybridization chamber (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 the results of hybridization.

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.

For loci corresponding to exon 19 and codons 719, 790, and 858 of the EGFR gene, to donors 13 and 61 of the KRAS gene, and codon 600 of the BRAF gene, a fluorescent signal is observed only in immobilized oligonucleotides corresponding to the wild-type sequence. In oligonucleotides corresponding to the 12 codon of the KRAS gene, a fluorescent signal is observed only in the oligonucleotide corresponding to the somatic mutation G12C. Therefore, the analyzed sample contains the mutation G12C KRAS.

Claims (4)

1. The method of analysis of somatic mutations in the genes EGFR, KRAS and BRAF, comprising the following stages:
(a) - amplification of fragments of the EGFR, KRAS and BRAF genes using LNA-blocking multiplex "nested" PCR, in which a tumor DNA sample is used as an amplification matrix; during PCR, a set of primers is used with the sequences shown in SEQ ID NO: 1-25 and a set of LNA oligonucleotides shown in SEQ ID NO: 26-32; as a result, a predominantly single-chain fluorescently labeled PCR product is formed;
(b) providing a biochip for identifying somatic mutations in the EGFR, KRAS and BRAF genes containing a set of immobilized oligonucleotides with the sequences shown in SEQ ID NO: 33-72;
(c) hybridization of a fluorescently labeled PCR product obtained in stage (a) on a biochip obtained in stage (b);
(d) - registration and interpretation of hybridization results on a biochip carried out at stage (c). 2. The method according to p. 1, in which the registration of the results of hybridization in stage (d) is carried out using a device capable of recording and recording fluorescence signals, equipped with special software that allows for 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 EGFR, KRAS and BRAF genes amplified using the set of primers and LNA oligonucleotides described in claim 1, a biochip is used, which is a substrate with a set of oligonucleotides immobilized on it with the sequences presented in SEQ ID NO: 33-72. 4. A set of oligonucleotide probes used to obtain a biochip used to identify somatic mutations in the EGFR, KRAS and BRAF genes, the probes having the sequences shown in SEQ ID NO: 33-72.

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