RU2631824C1 - Method for identification of braf gene mutations in human tumours - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: BRAF gene fragment under investigation is amplified by asymmetric real-time PCR with two TaqMan probes: BRAF-15-sense - 5'-ROX-aggtgattttggtctagctacagtgaaatc-BHQ2 and BRAF-15-antisense - 5'-Cy5-acccactccatcgagatttcactgta-BHQ2 and primers: direct - 5'-gagatctactgttttcctttactt, reverse - 5'-ggccaaaaatttaatcagt. The stage of primers and probes annealing is 2 s, the number of amplification cycles is 80-100, Taq polymerase concentration is 0.63 units, the annealing temperature of primers and probes is 57°C. Further, the probe-single-stranded DNA duplex melting and construction of melting peaks are performed. Mutations are identified by asymmetry of melting peaks.

EFFECT: method has a high sensitivity, 10 to 15 times higher than the prototype sensitivity, is simple in design, implemented for 2 hours in a single tube with single results recording, reduces the risk of samples cross-contamination.

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Description

The invention relates to medicine, namely to genetics, molecular diagnostics and oncology, and relates to a method for detecting gene mutations in human tumors for the use of targeted therapy.

In many oncological diseases, in particular with lung cancer, colon, melanoma, the obligatory objects for detecting gene mutations are the genes KRAS, NRAS, BRAF. For this purpose, Sanger sequencing is used - the “gold standard” of clinical genodiagnosis.

The disadvantages of the method: low sensitivity, complexity, the risk of cross-contamination of clinical samples.

A known method for detecting gene mutations by DNA melting (DNA Melting Analysis, DMA), sometimes implemented in high resolution mode (High Resolution Melting Analysis, HRMA) (Erali and Wittwer, High resolution melting analysis for gene scanning. Methods, 2010, 50, 250 -261; Krypuy et al., High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer (BMC Cancer, 2006, 6, 295).

The disadvantages of the method: relatively low sensitivity, the need for expensive equipment.

A known method for detecting gene mutations using PCR blocking agents - peptide nucleic acids 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 Clamp Improves Detection of T790m Mutation. PLoS ONE, 2013, 8, e67782).

The disadvantages of the method: the need for independent identification of the mutant allele, expensive chemical synthesis.

The prototype of the proposed method is a method for detecting gene mutations by DMA using TaqMan probes (Huang et al., Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes. PLoS ONE, 2011, 6, e19206). The prototype includes the following techniques: a) isolation of DNA from a sample of tumor tissue; b) amplification of a specific gene fragment in asymmetric real-time PCR using a TaqMan fluorescence probe; c) the use of the TaqMan probe also at the stage of DNA melting. Characteristic changes in the melting peaks of the probe duplexes with single-stranded DNA make it possible to detect a mutation in the analyzed sequence.

The disadvantage of this method is the low sensitivity of detecting gene mutations in small tumor clones that determine the drug resistance of the tumor.

The objective of the invention is the creation of a new, more sensitive way to detect gene mutations of BRAF in human tumors.

Technical result: the inventive method has a high sensitivity for detecting BRAF gene mutations, which is 10 or more times the sensitivity of the prototype, while maintaining ease of implementation, implementing a 2-hour test in one tube without intermediate or additional procedures with a single recording of results and without the risk of cross-contamination of samples DNA of the surgical material and tumor tissue enclosed in paraffin blocks.

The essence of the proposed method consists in detecting BRAF mutations in the tumor DNA associated with tumor sensitivity to targeted therapy using real-time asymmetric PCR with two TaqMan fluorescent probes used as agents that block the synthesis of wild-type alleles and post-amplification duplex melting single-stranded DNA probe. Analysis of the melting results is carried out using a standard melting module (DNA Melting Analysis) of a PCR apparatus (Bio-Rad CFX-96).

The oligonucleotides hybridized with the matrix located along Taq polymerases, in particular TaqMan probes, slow down the enzyme (Montgomery et al., The Influence of Nucleotide Sequence and Temperature on the Activity of Thermostable DNA Polymerases. The Journal of Molecular Diagnostics, 2014, 16, 305-313). Under specially selected PCR conditions, TaqMan probes suppress the amplification of the wild-type allele more strongly than the mutant allele due to the unpaired base in the latter case, which significantly weakens the structure of the duplex. Due to the preferential amplification of the mutant allele, the sensitivity of the method significantly increases.

The inventive method includes the following steps: amplification of the studied fragment of the BRAF gene by asymmetric real-time PCR with two TaqMan probes (sense and antisense); single-stranded DNA duplex melting; building melting peaks with the built-in Bio-Rad CFX Manager. The method is carried out in one device and one test tube without operator intervention.

The inventive method is as follows.

a) Obtaining biological material - tumor tissue, frozen in liquid nitrogen or fixed with formaldehyde and enclosed in paraffin blocks.

b) Isolation of DNA from tumor tissue by deproteinization with phenol and chloroform, and from paraffin blocks - using the commercial QIAamp DNA FFPE Tissue Kit (Qiagen).

c) Amplification of the BRAF gene fragment in two PCR reactions, the first one synthesizes mainly the antisense strand of the amplicon (the ratio of forward and reverse primers is 1:15), the second the semantic strand of the amplicon (the ratio of forward and reverse primers is 15: 1), which allows detect BRAF mutations in both strands of amplicon. The reaction was carried out in 96-well plates on a CFX96 instrument (Bio-Rad Laboratories, USA). The incubation mixture (25 μl) contained 50 mM Tris-HCl, pH 8.8; 50 mM KC1; 0.01% Tween-20; 3 mM MgCl ₂ ; 0.25 mm each of deoxynucleoside triphosphates; 0.2 x EvaGreen (Biotium, USA); 0.1 mg / ml BSA; 1 mM DTT; primers: direct - 5'-gagatctactgttttcctttactt, reverse - 5'-ggccaaaaatttaatcagt (0.4 μM direct primer and 0.027 μM reverse primer - for the predominant synthesis of sense strand amplicon; 0.4 μM reverse primer and 0.027 μM direct primer - for predominant synthesis amplicon antisense strand); 0.25 μM of two TaqMan probes (BRAF-15-sense - 5'-ROX-aggtgattttggtctagctacagtgaaatc-BHQ2 and BRAF-15-antisense 5'-СУ5-acccactccatcgagatttcactgta-BHQ2), blocking the synthesis of both strands of amplicon wild 0.63 units Hot-rescue Taq polymerase; 5 μl of a solution of DNA in water. PCR conditions: initial denaturation - 5 min at t = 95 ° C, after which 80-100 cycles - 13 s at t = 95 ° C, 2 s at t = 57 ° C with registration of fluorescence. Both probes present in the incubation medium (sense and antisense) function as blocking agents that slow down the synthesis of both strands of the wild-type amplicon.

d) Melting of single-stranded DNA duplexes with TaqMan probes: the reaction products were denatured for 1 min at t = 95 ° C, heated for 2 min at t = 55 ° C, and then the temperature was raised to t = 85 ° C in increments of 0.4 ° C ( shutter speed 6 s); data were analyzed using the Bio-Rad CFX Manager software (version 1.6). Only one of the two TaqMan probes present in the incubation medium (complementary to the amplicon strand that was predominantly synthesized by PCR) is involved in DNA melting. The detection of mutations in both strands of amplicon increases the reliability and sensitivity of the analysis.

The duration of PCR and DNA melting is 2 hours.

The method is illustrated in FIG. 1 (A, B, C), 2 (A, B, C), 3 (A and B), 4 (A and B).

In FIG. 1 (A, B, C) presents the stages of detecting BRAF gene mutations by asymmetric real-time PCR followed by melting of DNA with TaqMan probes in a Bio-Rad CFX96 instrument: in FIG. 1 (A) - asymmetric real-time PCR with TaqMan probes (hydrolysis of the probe during the reaction is accompanied by “flare-up” of fluorescence, which allows real-time judging of the progress of the reaction); in FIG. 1 (B) - DNA melting leads to a phase transition of DNA from double-stranded to single-stranded form, which is accompanied by a decrease in fluorescence; in FIG. 1 (C) - melting peaks (negative first derivatives of the melting curves with respect to temperature): a symmetric peak, homoduplex (curve 1) indicates the presence of only the wild-type allele, while an additional peak on the left, heteroduplex (curve 2) indicates the presence of a mutant allele.

In FIG. 2 (A, B, C) presents a diagram of the proposed method for the primary amplification of mutant alleles using TaqMan probes as blocking agents. The Taq polymerase enzyme, which extends the primer on the DNA template, encounters a TaqMan probe hybridized with the template and is forced to eliminate it from its path using 5'-3'-exonuclease activity. In FIG. 2 (A) is a diagram of amplification of a wild-type allele, the direction of movement of Taq polymerase is indicated by an arrow; F - fluorophore at the 5'-end of the probe, Q (quencher) - quencher at the 3'-end of the probe; The TaqMan probe is fully complementary to the wild-type DNA sequence. In FIG. 2 (B) - amplification of the mutant allele; upon hybridization of the probe with mutant DNA, an unpaired base appears, sharply weakening the structure of the complex and facilitating the movement of Taq polymerase. In FIG. 2 (C) presents a comparative analysis of a sample of DNA of lung cancer according to the method of the prototype (1) and according to the claimed method (2). When applying the proposed method, there was a suppression of amplification of the wild-type allele (wt) and an increase in the proportion of the mutant allele (mut).

In FIG. 3 (A and B) presents the definition of sensitivity (detection limit of mutant BRAF alleles) by the prototype method and by the present method by serial dilution of RKO cell DNA (V600E mutation) in wild-type DNA. (A) a prototype; (B) - the claimed method. Additional melting peaks located to the left of the main one (wild type, wt) are due to the presence of mutant alleles, since the melting point of the heteroduplex is lower than the melting temperature of the homoduplex. Arrows indicate the percentage of mutant DNA in the sample. The detection limits of the mutant V600E allele of the prototype and the claimed method are 6.3% and 0.4%, respectively. It is shown that the sensitivity of the proposed method is 15 times higher than the sensitivity of the prototype method.

In FIG. 4 (A and B) presents comparative results of the detection of mutations according to the prototype and the claimed method of DNA samples of lung cancer (A) and melanoma (B). Peaks of melting: 1 - prototype; 2 - the claimed method. The arrows indicate the sign identifying the mutation - the asymmetry of the melting peak on the left, detected only by the claimed method.

Claims (1)

A method for detecting BRAF mutations in human tumors, including amplification of the studied gene fragment in the selected conditions of asymmetric real-time PCR; single-stranded DNA duplex melting; building melting peaks; identification of mutations according to the asymmetry of the melting peaks, characterized in that two selected TaqMan probes are used as blocking agents: BRAF-15-sense - 5'-ROX-aggtgattttggtctagctacagtgaaatc-BHQ2 and BRAF-15-antisense - 5'-Сu5-acccacttact2 and primers: direct - 5'-gagatctactgttttcctttactt, reverse - 5'-ggccaaaaatttaatcagt, the stage of annealing of primers and probes is 2 s, the number of amplification cycles is 80-100, the concentration of Taq polymerase is 0.63 units, the selected temperature of annealing of primers and probes is 57 ° C.

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