RU2451086C1 - Method for detecting specific nucleotide sequences and nucleotide replacements by real-time pcr with effect of probe fluorescence quenching shown by primer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves nucleic acid recovery from analysed samples. Related oligonucleotide probes and primers are sampled, and PCR-amplification of target nucleic acid follows. Probe melting curves are analysed. Amplification of target nucleic acid is ensured by a relevant pair of primers, one of which, namely reverse, serves to form a DNA chain, complementary to the probe, and contains a non-fluorescent fluorescence quencher inside a nucleotide sequence, is closer to the 3' terminal. Detecting a site of target nucleic acid found between binding sites of primers is ensured by using specified 3'-fluorescent marked oligonucleotide probe a binding site of which is partially blocked with a determinant of an unmarked forward primer on one side, and on the other side it is spaced at a number of nucleotides from the 3'-terminal of the primer carrying the fluorescence quencher.

EFFECT: invention provides more effective real-time PCR analysis of specific nucleotide sequences and nucleotide replacements.

12 cl, 10 dwg, 3 tbl, 3 ex

Description

The invention relates to the field of molecular biology, biotechnology, genetic engineering and medical molecular diagnostics, gene diagnostics, namely the identification of specific nucleotide sequences and nucleotide substitutions in known nucleic acid sequences using the real-time polymerase chain reaction method (PCR-RV) with the effect quenching of fluorescence probe with primer. The invention can be used to detect specific nucleotide sequences, nucleotide substitutions (polymorphisms) in known nucleotide sequences and other natural or engineering gene mutations in a complex genetic environment.

Various methods are known for detecting specific nucleotide sequences and nucleotide substitutions in the studied nucleic acid sequences using real-time polymerase chain reaction (PCR-RV) with fluorescently-labeled oligonucleotide probes. The most widely used 5'-exonuclease method, based on the effect of spatial separation of the fluorophore and fluorescence quencher due to Taq cleavage with the DNA polymerase of the probe associated with the amplified DNA sequence (Heid, C. A., J. Stevens, K. J. Livak, and R. M. Williams. 1996. Real time quantitative PCR. Genome Res. 6: 986-994).

The main disadvantages of this method:

1) The need to use separate probes for each detected nucleotide sequence (or nucleotide substitution) and the consequent limitation on the possibility of simultaneous detection of multiple sequences (or variants of one sequence) in one reaction. Moreover, the maximum number of simultaneously determined sequences corresponds to the number of fluorescently-labeled probes used, the signal of which can be separated by a detecting device.

2) The inability to identify new (unknown) nucleotide polymorphisms in a given section of the sequence.

There are alternative PCR-PB methods that can partially overcome the above limitations by using probe melting curve analysis (Lyon, E. 2001. Mutation detection using fluorescent hybridization probes and melting curve analysis. Expert. Rev. Mol. Diagn. 1: 92-101) after amplification is completed. These include: PCR-RV with paired probes with resonant energy transfer (FRET) (Cardullo, RA, S. Agrawal, C. Flores, P. C. Zamecnik and DEWolf. 1988. Detection of nucleic acid hybridization by nonradiative fluorescence Resonance energy transfer. Proc. Natl. Acad. Sci. USA 85: 8790-8794. Didenko, VV 2001. DNA probes using fluorescence resonance energy transfer (FRET): designs and applications. Biotechniques 31: 1106-1), PCR-RV with resonant energy transfer between the non-specific SYBR Green I DNA-binding fluorophore and the fluorescently-labeled probe (iFRET) (Howell, WM, M. Jobs, and AJBrookes. 2002. iFRET: an improved fluorescence system for DNA-melting analysis. Genome Res . 12: 1401-1407), PCR-RV with probes containing complementary terminal sequences Molecular Beacons (Tyagi, S. and FRKramer. 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14: 303-308), PCR-RV with probes containing a DNA ligand and a fluorescence quencher on 5'-end and fluorophore at the 3'-end (MGB Eclipse) (Afonina, IA, MWReed, E. Lusby, IGShishkina, and YSBelousov. 2002. Minor groove binder-conjugated DNA probes for quantitative DNA detection by hybridization-triggered fluorescence. Biotechniques 32: 940-949) and PCR-RV with fluorescence energy transfer between donor and acceptor fluorophores located on the probe and primer (Lay, MJ and C. T. Wittwer. 1997. Real-time fluorescence genotyping of factor V Leiden during rapid -cycle PCR. Clin. Chem. 43: 2262-2267. Wittwer, S.T., Rasmussen, RP, and Lay, M. Monitoring amplification with FRET probes. University of Utah Research Foundation, US. (US Patent 7160998). 09-01-2007). In all these cases, the analysis of melting curves involves the use of probes whose fluorescence differs in the free and bound state (with the target DNA). The analysis is carried out by recording fluorescence (F) with a gradual increase in temperature (T) of the mixture in which the probe and complementary DNA are located. When the probe binds to a fully complementary sequence, a duplex with the highest melting point (Tm) is formed, while the binding of the probe with a partially mismatching (mutant) sequence leads to the formation of a less stable complex with a lower Tm. Thus, the analysis of the fluorescence curve of the probe depending on the temperature of the reaction mixture (dF / dT) allows one to determine the Tm values of duplexes and identify DNA sequences that are both completely complementary to the probe and differing in single nucleotide substitutions. It is possible to simultaneously determine a fully complementary and mutant sequence (for example, heterozygosity analysis) using one probe. The main disadvantage of the above approaches is the difficulty in detecting certain nucleotide substitutions in variable DNA sequences, where mutations of interest are surrounded by other (satellite) polymorphisms. The use of all these PCR-RV variants is fundamentally impossible in all cases when the minimum length of the probe or its fragment involved in binding to the target DNA exceeds the distance between satellite polymorphisms.

The closest analogue of the invention can be considered the aforementioned PCR-RV method with the transfer of fluorescence energy between donor and acceptor fluorophores located on the probe and primer (Lay, MJ and C. T. Wittwer. 1997. Real-time fluorescence genotyping of factor V Leiden during rapid -cycle PCR. Clin. Chem. 43: 2262-2267). In this case, as with classical FRET probes (Didenko, VV 2001. DNA probes using fluorescence resonance energy transfer (FRET): designs and applications. Biotechniques 31: 1106-1), pairs of fluorescent dyes are used ( donor and acceptor) between which resonance fluorescence energy transfer is possible (Forster. 1948. Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann. Physik. 437: 55). The principal disadvantages of both methods are as follows:

1) The possibility of partial cleavage of probes during the NDP due to the 5'-exonuclease activity of DNA polymerase.

2) The impossibility of conducting multiplex analysis for the simultaneous detection of several DNA sequences in one reaction due to the limited set of pairs of donor and acceptor dyes that differ in fluorescence spectra.

The disadvantages of the described approaches are eliminated in the present invention. Based on the foregoing, the objective of the present invention is to increase the efficiency and expand the capabilities of the analysis of specific nucleotide sequences and nucleotide polymorphisms using the PCR-RV method through the use of a new approach based on the quenching of fluorescence of the probe with a primer carrying a non-fluorescent fluorescence quencher.

The essence of the invention lies in the fact that a method for detecting specific nucleotide sequences and nucleotide substitutions using real-time PCR with the effect of quenching the fluorescence of the probe with a primer includes the selection of nucleic acids from the samples to be studied, the selection of appropriate oligonucleotide probes and primers, PCR amplification of one or more necessary (target) sections of the nucleotide sequence, analysis of the melting curves of the probes after PCR, while for the amplification of each of the target The nucleotide sequence astringes use the corresponding pair of primers, one of which, namely, a reverse primer, serves to form a DNA strand complementary to the probe and contains a non-fluorescence fluorescence quencher inside the nucleotide sequence, closer to the 3 'end, and to detect the target nucleotide sequence located between sites of primer binding, use the appropriate 3'-fluorescently-labeled oligonucleotide probe, the binding site of which on one side is partially overlapped is associated with the binding region of the unlabeled (forward) primer, and on the other hand is located at a distance of several nucleotides from the 3'-end of the primer carrying the fluorescence quencher, so that the binding of the probe to the DNA chain formed as a result of elongation of the reverse primer leads to a convergence fluorophore and quencher at a distance sufficient to effectively quench fluorescence due to fluorescence resonance energy transfer (FRET) or the formation of stable complexes between the fluorophore and quencher (static silt quenching contact).

There are a number of embodiments of the proposed method.

The second of them is that the detected specific nucleotide sequences are invariant, that is, they do not contain nucleotide substitutions, deletions, insertions and other gene mutations.

The third option is that, unlike the second option, it is a method for detecting nucleotide substitutions, deletions, insertions, and other point genetic polymorphisms (in the first nucleic acid compared to the second nucleic acid).

In a fourth embodiment of the method, the accumulation of PCR products is detected by decreasing the initial level of fluorescence of the probe directly during amplification.

In a fifth embodiment of the method, the sequences of the probe and primers are selected so that the Tm of the probe is 3-5 ° C higher than the Tm of the primers, and the overlap of the sequence of the probe and unlabeled primer is minimal, 1-3 nucleotides.

In a sixth embodiment of the method, the size of the amplified portion of the nucleotide sequence is 45-100 bp.

In a seventh embodiment of the method, the length of the overlapping portion of the probe and unlabeled primer sequences is one or more nucleotides.

In an eighth embodiment of the method, the sequences of the probe and the reverse primer carrying the quencher are selected such that the probe is bound to the target DNA at a distance of 0-20 nucleotides between the 3 'ends of the probe and the primer.

In a ninth embodiment of the method, a portion of a target nucleotide sequence complementary to a portion of a probe that does not overlap with an unlabeled forward primer comprises one or more nucleotide positions to be analyzed.

The tenth embodiment of the method involves the possibility of binding an unlabeled forward primer to the target DNA immediately before the analyzed nucleotide position, where satellite polymorphisms - mismatched nucleotides - may be present.

The eleventh embodiment of the method suggests the possibility of satellite polymorphisms: nucleotide substitutions, short deletions, insertions in the region of the nucleotide sequence between the 3 'end of the probe and 3' end of the reverse primer.

The twelfth embodiment of the method is that real-time PCR is real-time multiplex PCR.

The PCR-PB design described above allows the identification of various mutations in the binding region of the probe by analysis of the melting curves after amplification is completed. With complete match between the probe sequences and the target DNA, the Tm of the duplex is maximum, and in the presence of mismatched nucleotides it decreases to a different extent depending on the position and nature of the mutation.

The described method differs from the closest analogue (Lay, M.J. and C.T. Wittwer. 1997. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. Clin. Chem. 43: 2262-2267) in the following key ways:

1) Not acceptor fluorophores are used for primer labeling, but non-emitting fluorescence absorbers, such as: BHQ1 (Black Hole Quencher-1, λ _max abs = 534 nm, quenching operating range 480-580 nm), BHQ2 (Black Hole Quencher-2, λ _max abs = 579 nm, extinction range 560-670 nm), BHQ3 (Black Hole Quencher-3, λ _max abs = 672 nm, extinction range 620-730 nm), RTQ1 (Real Time Quencher-1, λ _max abs = 520 nm, the extinction range 470-570 nm), RTQ2 (Real Time Quencher-2, λ _max abs = 625 nm, the extinction range 580-670 nm) and other “dark quenchers”. The advantage of the BHQ and RTQ series of absorbers is that, having similar physical properties, they have different spectral characteristics and, therefore, can be used in combination with various fluorophores, including for multiplex detection. Preferred fluorophores that can be combined with quenchers of the BHQ and RTQ series are, for example, xanthine dyes, including fluoresceins, cyanines, rhodamines (Cook, RM, Lyttle, M., and Dick, D. Dark quenchers for donor-acceptor energy transfer. Biosearch Technologies, Inc. (US Patent 7109312). 19-9-2006. Haugland, RP, MTZ Spence, and ID Johnson. 1996. Handbook of Fluorescent Probes and Research Chemicals. Molecular Probes, Eugene, OR). Thus, our proposed method provides wide possibilities for the selection of donor-acceptor pairs (fluorophore-quencher) and multiplex analysis (simultaneous detection of several DNA targets in one reaction). Amplification products are detected by monitoring the fluorescence of the dye associated with the probe. The accumulation of PNR products during the amplification reaction can be judged by the decrease in the level of fluorescence (if the signal is recorded at a temperature below Tm of the probe-DNA duplex target). During post-amplification analysis of melting curves, the dissociation of the probe-DNA complex is accompanied by a sharp increase in fluorescence.

2) Partial overlapping of the binding sites of the probe and unlabeled primer is used, which eliminates the possibility of cleavage of the probe by polymerase during primer elongation, and the design of probes for detecting mutations in a complex genetic environment is facilitated. The close arrangement of the primers also provides high PCR efficiency.

The invention is illustrated by drawings.

In figure 1. The general arrangement of primers and probe is presented, where 1 is a probe, 2 is a primer, 3 is a sequence interruption site, 4 is a fluorophore, 5 is a fluorescence quencher.

In figure 2. the binding of the probe to the complementary DNA strand is presented.

Figure 3 presents the original sequence containing flanking satellite polymorphisms, where 6 are satellite polymorphisms, 7 is the analyzed nucleotide position.

Figure 4 shows the binding of the primer to the original sequence.

Figure 5 presents the NDP product.

Figure 6 shows the binding of the probe.

Figure 7 presents an analysis of the melting curves in the case of quenching the fluorescence of the probe upon binding to a complementary DNA chain, for a PNR product containing a nucleotide substitution, 8, a PNR product completely complementary to the probe, 9, and for an indicative sample in the absence of PCR -product - 10, where F is fluorescence, T is temperature, dF / dT is the derivative of fluorescence with respect to temperature, Tm1 is the melting temperature of the probe upon binding with a fully complementary sequence, Tm2 is the melting temperature of the probe upon binding with the mutant sequence lnostyu.

On Fig. an example of mutation detection is presented: Glu104Lys, Arg164Cys / His / Ser, Ala237Thr, Gly238Ser and Glu240Lys, which determine the extended TEM β-lactamase activity spectrum using multiplex PCR-RV with the effect of quenching the fluorescence of the probe with a primer and analyzing the melting curves, X, where F5, where F , R6G - dyes.

In Fig.9. presents an example of mutation detection: Ser83Phe, Asp87Gly / Tyr / Asn in QRDR GyrA, which determine the quinolone resistance in Salmonella enterica using PCR-RV with the effect of quenching probe fluorescence by primer and analysis of melting curves.

10. an example of detection of allelic variants of VKORC1 and CYP2C9 associated with hypersensitivity to warfarin using multiplex PCR-RV with the effect of quenching probe fluorescence by primer and analysis of melting curves is presented.

The implementation of the invention

The method is as follows:

1) Carry out the selection of sequences of primers 1 (1, 2, 4, 5, 6) and probes 2 (1, 2, 6) in accordance with the standard rules described in the literature (Peters, IR, C.R. Helps, EJHall, and MJDay. 2004. Real-time RT-PCR: considerations for efficient and sensitive assay design. J.Immunol.Methods 286: 203-217) and taking into account the following specific requirements:

a) first, the choice of probe 2, complementary to the analyzed DNA sequence. Then amplification primers 1 are selected so that the 3 'end of primer 1 carrying the fluorescence quencher 3 (Figs. 1, 2, 5, 6) is at a distance of 0-10 nucleotides from the probe binding site, and 3' and 5 'ends unlabeled primer and probe partially overlapped;

b) if it is necessary to register the accumulation of PCR products directly during amplification (for example, for quantitative analysis of the target DNA in the studied samples), it is recommended to select the probe and primer sequences so that the probe Tm is 3-5 ° C higher than the Tm primers and the overlap of the sequence of the probe and unlabeled primer 4 (Figs. 1, 2, 4, 5, 6) was minimal and amounted to 1-3 nucleotides;

c) in the absence of the need to register the accumulation of PCR products during amplification of Tm probe and primers can be approximately the same and can vary over a wide range;

d) to identify specific nucleotide substitutions, the selection of a DNA strand complementary to the probe is recommended so that the values of Tm of the probe upon binding to a fully complementary and mutant sequence differ as much as possible.

2) Labeled oligonucleotide primers and probes are synthesized by chemically attaching fluorophore 5 (FIGS. 1, 2, 6) or a quencher to an oligonucleotide sequence selected in accordance with the requirements of the method. When creating the probe of this invention, the fluorescent label is preferentially attached to the 3'-end of the oligonucleotide sequence, although attachment to internal sites can also be used in some specific cases. When creating labeled primer 4, in accordance with this invention, the fluorescence quencher 3 is attached to the internal position of the oligonucleotide chain. Fluorophores 5 (or quenchers 3) are introduced into the oligonucleotide probe 2 (or primer 1) mainly in the form of amidite derivatives. Alternatively, fluorophores 5 (or other low molecular weight labels) containing reactive moieties (e.g., isothiocyanates or activated esters) can be introduced by reaction with a reactive moiety of a linker attached to a nucleic acid (e.g., hexylamine). In addition, fluorophore 5 can be attached to the 3'-end of the oligonucleotide sequence using synthesis on a modified support (derivatized) solid support (Cook, RM, Lyttle, M., and Dick, D. Dark quenchers for donor- acceptor energy transfer. Biosearch Technologies, Inc. (US Patent 7109312). 19-9-2006).

3) Nucleic acids are isolated using any standard methods for subsequent PNR analysis. The presence of potential PCR inhibitors in the samples has a minimal effect on the amplification results using the described approach compared to other PCR-PB variants due to the short length of the PNR products. If the studied samples have a high initial concentration of the target DNA / RNA (> 10 ³ -10 ⁴ copies per reaction), it is possible to use methods of rapid cell lysis (using elevated temperature, alkaline and chaotropic reagents, etc.) without subsequent removal of inhibitors .

4) Carry out a DNA amplification reaction using real-time PCR instruments. An equimolar ratio of primers 1 may be used in the reaction, however, asymmetric PCR with a ratio of unlabeled and labeled primers of 1: 1: 2-1: 10 is recommended. An excess of primer 1 carrying a fluorescence quencher 3 promotes preferential accumulation of a DNA strand complementary to probe 2 during PCR, which ensures maximum detection efficiency. Recommended concentrations of primers 1 in the PCR mixture: 40-800 nM; probe concentration 2: 80-400 nM. In the case of asymmetric PCR, the number of amplification cycles can be increased compared to conventional PCR. When conducting a quantitative PCR analysis, the fluorescence level is measured at the stage of annealing of primers 1. Due to the short length of the PCR products, the duration of the stages of annealing and elongation of primers 1 can be reduced to several seconds.

5) After amplification is completed, the melting curves are analyzed (FIG. 3). The spatial separation of fluorophore 5 and quencher 3 as a result of melting of the probe-DNA-target complex leads to an increase in fluorescence. Identification of amplification products or detection of nucleotide substitutions in the binding region of the probe is carried out according to the values of Tm corresponding to the peaks of the melting curves in the graph dF / dT vs T.

Example 1. Multiplex detection of mutations in TEM genes of β-lactamases, which determine the extended spectrum of resistance to β-lactam antibiotics in clinical strains of the Enterobacteriaceae family

TEM-type β-lactamases are one of the most common enzymes that break down β-lactam antibiotics and cause their resistance to gram-negative bacteria. The point mutations in the bla _TEM genes corresponding to the amino acid substitutions Glul04 → Lys, Arg164 → Cys / His / Ser, Ala237 → Thr, Gly238 → Ser and Glu240 → Lys determine the extended spectrum of TEM enzymes activity - the ability to hydrolyze all modern antibiotics of the cephalosporin group.

To detect 7 nucleotide substitutions in the PCR-PBX multiplex format, three pairs of primers flanking the mutation sites in codons 104, 164, 237-240, three probes complementary to the initial (non-mutant) bla _TEM-1 sequence labeled, respectively, FAM, R6G, ROX, and one probe complementary to the mutant Lys240 variant labeled with Cy5 (Table 1, which shows oligonucleotide primers and probes used to detect mutations in TEM β-lactamase genes).

The composition of the PCR mixture with a total volume of 25 μl: primers and probes in the concentration indicated in table 1; 0.2 mM dNTP; 2 mM MgCl ₂ ; 2.5 units Dia Tak DNA polymerase (LLC InterLabService, Russia); 67 mM Tris-HC1 (pH = 8.3); 17 mM (NH ₄ ) ₂ SO ₄ ; 0.1% Tween-20; 0.12 mg / ml bovine serum albumin (BSA); 8% glycerol and 5 μl of DNA sample. To ensure a “hot start” of PCR, a mixture of primers, probes, and dNTP was added to the reaction tubes separately, covered with a layer of low-melting wax, and after it solidified, mixtures of other components were added.

Isolation of bacterial DNA: 2-3 colonies of each culture were resuspended in 100 μl of TE buffer; incubated for 20 min at 99 ° C; centrifuged for 2 min at 10,000 g; 5 μl of supernatant was used in PCR.

Amplification and analysis of the melting curves was carried out using the Rotor-Gene 2000 system according to the following protocol: initial incubation for 15 minutes at 80 ° C; 1 min denaturation at 95 ° C; then 40 cycles of 15-second denaturation at 95 ° C and 15-second annealing-elongation at 61 ° C with fluorescence detection on JOE and Cy5 channels; analysis of melting curves with an initial incubation of 2 min at 45 ° C and a subsequent increase in temperature by 1 ° C every 10 seconds to 80 ° C with constant detection of fluorescence on the FAM, JOE, ROX and Cy5 channels.

Identification of wild type bla _TEM sequences and mutations in codons 104, 164 and 237-240 was carried out in accordance with the melting temperature of the probes (figure 4). Enterobacteriaceae strains with the known nucleotide sequence of bla _TEM genes were used as controls, including 5 cultures with wild-type genes that differ only in the spectrum of silent mutations, and 14 cultures with mutations corresponding to the amino acid substitutions Glu104 → Lys, Arg164 → Cys / His / Ser / Ser , Ala237 → Thr, Gly238 → Ser, Glu240 → Lys. The method was also used to screen the listed mutations in 718 clinical strains of enterobacteria-causative agents of nosocomial infections.

For control samples, the possibility of detection and differentiation of all the above mutations in any combination using multiplex PCR-RV and analysis of the melting curves of the probes is shown. Differences in the values of Tm probes upon binding to fully complementary and mutant target DNA sequences ranged from 4 to 10 ° C (Fig. 4). The possibility of detecting key mutations (for example, mutations in codon 164) was demonstrated regardless of the presence of satellite polymorphisms 6 located in the immediate vicinity (Fig. 3) (flanking mutations in codons 160 and 165). When analyzing clinical strains in 533 (74.2%) cultures, wild-type bla _TEM sequences were found, in 1 (0.1%) strain, G / A (Arg164His) substitution was confirmed, the presence of which was confirmed by gene sequencing.

Example 2. Analysis of mutations of resistance to quinolones in Salmonella enterica

Fluorinated quinolones are the main antimicrobial agents for the treatment of invasive salmonella. The resistance of salmonella to quinolones is associated with target mutations, A of the DNA gyrase subunit (GyrA), in the region of amino acid residues 83-87, which participate in the binding of quinolones and form the so-called QRDR site (Quinolone Resistance Determining Region).

The PCR-RV method with the effect of quenching the fluorescence of the probe with a primer was used to simultaneously detect nucleotide substitutions corresponding to the most frequent mutations: Ser83 → Phe, Asp87 → Gly / Tyr / Asn, causing quinolone resistance. The reaction used one pair of primers flanking the QRDR region of gyrA, and one probe, the sequence of which coincides with the non-mutant QRDR variant (Table 2, which shows the oligonucleotide primers and the probe used to detect mutations in QRDR gyrA).

The composition of the PCR mixture with a total volume of 25 μl: primers and probes in the concentration indicated in table 2; 0.2 mM dNTP; 2 mM MgCl ₂ ; 2.5 units TaqF DNA polymerase (LLC InterLabService, Russia); 67 mM Tris-Hcl (pH = 8.3); 17 mM (NH ₄ ) ₂ SO ₄ ; 0.1% Tween-20; 0.12 mg / ml bovine serum albumin (BSA); 8% glycerol and 3 μl of DNA sample.

Isolation of bacterial DNA: 2-3 colonies of each culture were resuspended in 100 μl of TE buffer; incubated for 20 min at 99 ° C; centrifuged for 2 min at 10,000 g; 3 μl of supernatant was used in PCR.

Amplification and analysis of the melting curves was carried out using the Rotor-Gene 2000 system according to the following protocol: initial denaturation 15 min at 95 ° C; then 45 cycles of 15-second denaturation at 95 ° С and 20-second annealing-elongation at 55 ° С with fluorescence detection on the FAM channel; analysis of melting curves with an initial incubation of 3 min at 35 ° C and a subsequent increase in temperature by 1 ° C every 10 seconds to 75 ° C with constant detection of fluorescence on the FAM channel.

Mutations were identified in accordance with the probe melting temperature: 59.2 ± 0.05 ° С for the wild-type QRDR sequence, 53.8 ± 0.14 ° С for the Gly83 / Tyr83 / Asn83 and 50.7 ± 0 variants 16 ° C for Phe83 (Fig. 5). Strains of S. enterica Typhimurium serotype with known mutations in QRDR gyrA were used as controls. The method was used to analyze the structure of QRDR in 48 clinical Salmonella strains that are sensitive to quinolones and 32 isolates resistant to nalidixic acid (minimum inhibitory concentration [IPC] ≥128 mg / L). In sensitive strains, no mutations in QRDR were detected; mutations at position 87 were found in 25 resistant isolates; in seven, the replacement was Ser83 → Phe. The analysis results were confirmed by sequencing of the gyrA site in 100% of cases.

Example 3. Identification of genetic markers of hypersensitivity to oral anticoagulants in humans

Oral anticoagulant drugs of the coumarin group (warfarin) are widely used in clinical practice for the prevention of arterial and venous thrombosis. The pharmacokinetics and pharmacodynamics of warfarin are extremely variable and depend on many factors, including genetically determined differences in metabolic rate under the action of cytochrome P450 encoded by the CYP2C9 gene, and differences in the expression level of the VKORC1 gene encoding the warfarin target - vitamin K-epoxide reductase. Allelic variants CYP2C9 * 2 (-485Т → А; -484С → А), CYP2C9 * 3 (42614А → C) and VKORC1 * 2 (6484С → T) are associated with hypersensitivity to warfarin. Identification of these genetic markers is recommended in order to reduce the risk of overdose and bleeding during warfarin therapy in all patients.

To detect the listed polymorphisms 6 in the PCR-PBX multiplex format, three pairs of primers were used that flanked the mutation sites at -485 / -484, 42614 CYP2C9 and 6484 VKORC1, and three probes complementary to the non-mutant sequences CYP2C9 * 1 and VKORC1 * , respectively, FAM, R6G and ROX (table 3, which shows the oligonucleotide primers and probes used to detect allelic variants of VKORC1 and CYP2C9 associated with hypersensitivity to warfarin).

The composition of the PCR mixture with a total volume of 25 μl: primers and probes in the concentration indicated in table 3; 0.2 mM dNTP; 2 mM MgCl ₂ ; 2.5 units TaqF DNA polymerase (LLC InterLabService, Russia); 67 mM Tris-Hcl (pH = 8.3); 17 mM (NH ₄ ) ₂ SO ₄ ; 0.1% Tween-20; 0.12 mg / ml bovine serum albumin (BSA); 8% glycerol and 5 μl of DNA sample.

DNA was isolated from 100 μl of whole blood using commercial DNA-sorb-B kits (OOO InterLabService, Russia).

Amplification and analysis of the melting curves was carried out using the Rotor-Gene 2000 system according to the following protocol: initial denaturation 15 min at 95 ° C; then 55 cycles of 15-second denaturation at 95 ° С, 10-second annealing at 58 ° С with fluorescence detection on FAM, JOE and ROX channels and 10-second elongation at 72 ° С; analysis of melting curves with an initial incubation of 3 min at 45 ° C and a subsequent increase in temperature by 1 ° C every 10 seconds to 80 ° C with constant detection of fluorescence on the FAM, JOE and ROX channels.

Allelic variants of CYP2C9 and VKORC1 were identified according to the melting temperature of the probes: CYP2C9 * 3-Pb3: 57.6 ± 0.10 ° C for CYP2C9 * 1 and 50.3 ± 0.11 ° C for CYP2C9 * 2; CYP2C9 * 2-Pb-485: 66.8 ± 0.15 ° C for CYP2C9 * 1 and 59.7 ± 0.21 ° C for CYP2C9 * 3; VKORC1 * 2-Pb: 62.6 ± 0.23 ° C for VKORC1 * 1 and 54.4 ± 0.29 ° C for VKORC1 * 2 (Fig. 6). As controls, we used DNA samples of persons of both sexes with known genotypes: CYP2C9 * 1 * 1, CYP2C9 * 2 * 2, CYP2C9 * 3 * 3, CYP2C9 * 1 * 2 and CYP2C9 * 1 * 3, VKORC1 * 1 * 1, VKORC1 * 2 * 2 and VKORC1 * 1 * 2. The method is also used to identify markers of hypersensitivity to warfarin in 42 patients requiring indirect anticoagulants. Polymorphisms 6 in the CYP2C9 and VKORC1 genes were found in 26 (62%) patients. In 6 (14%) patients, genotypes were identified suggesting hypersensitivity to warfarin and the need to reduce the starting dose of the drug: CYP2C9 * 1 * 1 / VKORC1 * 2 * 2 (n = 2), CYP2C9 * 1 * 2 / VKORC1 * 2 * 2 ( n = 2) and CYP2C9 * 1 * 3 / VKORC1 * 2 * 2 (n = 2).

The main advantage of the developed method is the ability to identify specific nucleotide sequences or to analyze specific nucleotide positions in a complex genetic environment. The design of an oligonucleotide probe for binding to a portion of a DNA sequence containing satellite polymorphisms 6 on either side of the nucleotide position of interest is usually difficult. In the proposed method, primer binding immediately before the nucleotide position of interest (in the region of one of the satellite polymorphisms 6) allows to obtain a PCR product with a constant sequence at the 5 'end. This allows you to select the probe of the required length, the sequence of which at the 5'-end overlaps with the sequence of the primer, and at the 3 'end it includes the analyzed nucleotide positions 7 (Fig.6). Nucleotide substitutions outside the binding region of the probe do not significantly affect the Tm probe-DNA duplex target.

Claims (12)

1. A method for detecting specific nucleotide sequences and nucleotide substitutions using real-time PCR with the effect of quenching the fluorescence of the probe with a primer, including: isolation of nucleic acids from the samples to be studied, selection of appropriate oligonucleotide probes and primers, PCR amplification of one or more necessary target nucleotide sites sequences, analysis of the melting curves of probes after PCR, characterized in that for amplification of each of the target sections of the nucleotide the sequences use an appropriate pair of primers, one of which, namely, a reverse primer, serves to form a DNA strand complementary to the probe and contains a non-fluorescent fluorescence quencher inside the nucleotide sequence, closer to the 3 'end, and to detect the target nucleotide sequence located between the binding sites primers use the appropriate 3'-fluorescence-labeled oligonucleotide probe, the binding site of which on one side partially overlaps with the binding region of unlabeled (forward) primer, and on the other hand is located at a distance of several nucleotides from the 3'-end of the primer carrying the fluorescence quencher, so that the binding of the probe to the DNA chain formed as a result of the elongation of the reverse primer leads to the convergence of the fluorophore and quencher a distance sufficient to effectively quench fluorescence due to fluorescence resonance energy transfer (FRET) or the formation of stable complexes between the fluorophore and quencher (static or contact quenching). 2. The method according to claim 1, characterized in that the detected specific nucleotide sequences are invariant, that is, do not contain nucleotide substitutions, deletions, insertions and other gene mutations. 3. The method according to claim 1, characterized in that it is a method for detecting nucleotide substitutions, deletions, insertions and other point genetic polymorphisms in the first nucleic acid compared to the second nucleic acid. 4. The method according to claim 1, characterized in that the accumulation of PCR products is recorded by reducing the initial level of fluorescence of the probe directly during amplification. 5. The method according to claim 4, characterized in that the sequence of the probe and primers is chosen so that the Tm of the probe is 3-5 ° C higher than the Tm of the primers, and the overlap of the sequence of the probe and unlabeled primer is minimal, 1-3 nucleotides. 6. The method according to claim 1, characterized in that the size of the amplified section of the nucleotide sequence is 45-100 bp. 7. The method according to claim 6, characterized in that the length of the overlapping portion of the probe sequences and unlabeled primer is one or more nucleotides. 8. The method according to claim 7, characterized in that the sequences of the probe and the reverse primer carrying the quencher are selected in such a way that the binding of the probe to the target DNA is carried out at a distance of 0-20 nucleotides between the 3 'ends of the probe and primer. 9. The method according to claim 3, characterized in that the portion of the target nucleotide sequence complementary to the portion of the probe that does not overlap with the unlabeled forward primer contains one or more nucleotide positions to be analyzed. 10. The method according to claim 9, characterized in that the binding of the unlabeled forward primer to the target DNA occurs immediately before the analyzed nucleotide position, and satellite polymorphisms of mismatched nucleotides may be contained in the binding site. 11. The method according to claim 3, characterized in that the portion of the nucleotide sequence between the 3 'end of the probe and the 3' end of the reverse primer contains satellite polymorphisms: nucleotide substitutions, short deletions, insertions. 12. The method according to claim 1, characterized in that the real-time PCR is a multiplex real-time PCR.

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