WO2018059525A1 - Composition, method and kit for detecting target nucleic acid sequence variant - Google Patents

Abstract

Provided is a composition or kit for detecting a target nucleic acid sequence variant. The kit comprises: a closed probe, a detection probe, and a primer; the closed probe is combined with a wild type variant of a target nucleic acid sequence by means of specific binding, and a tail end 3' thereof is modified with oligonucleotide for preventing its extension; the detection probe is combined with a mutant variant of the target nucleic acid sequence by means of specific binding and can generate a detection signal; the primer is a common primer for the wild type variant and the mutant variant of the target nucleic acid sequence. Also provided is a method for detecting a target nucleic acid sequence variant. The method achieves a purpose of high selectivity detection by enriching mutant variants of a target nucleic acid sequence in a nucleic acid sample by means of a PCR reaction, and finally detecting an amplicon according to changes in detection signals of a detection probe.

Description

Composition, method and kit for detecting target nucleic acid sequence variants

The present application claims priority to Chinese Patent Application No. 201610872112.5, entitled "A composition and method and kit for detecting target nucleic acid sequence variants", which is filed on September 30, 2016. The entire contents are incorporated herein by reference.

Technical field

The invention belongs to the field of molecular biology, and particularly relates to a composition and a method for detecting a variant of a target nucleic acid sequence, and the invention also relates to a kit for detecting a target nucleic acid variant, which is widely used for nucleic acid amplification, in vitro diagnosis of a genetic variant, Genotyping and other fields.

Background technique

A gene mutation is a change in gene structure caused by the addition, deletion or alteration of a base pair occurring in a DNA molecule. There are two types of gene mutations, sexual cell mutations and somatic mutations. Sexual cell mutation refers to a mutation that occurs in a sex cell and is a heritable mutation type. Mutations in somatic cells other than sex cells do not cause genetic changes in the offspring, but can cause changes in the genetic structure of some contemporary cells. Most somatic mutations have no phenotypic effects. Somatic mutation is a rare mutation. Somatic mutations are present in a large number of wild-type background DNA sequences, and the amount of somatic mutations is small relative to the content of the wild-type background sequence. For example, tumor tissue and peripheral blood contain a small amount of tumor cell DNA, and initial bacterial and viral resistance. Somatic mutations are often associated with the onset of disease and can serve as markers for disease onset, as a primary marker of prognosis, and as a marker for drug guidance. Therefore, the detection of somatic mutations is of great significance for the diagnosis and treatment of diseases and prognosis.

At present, the detection methods of somatic mutation mainly include DNA sequencing method, RFLP-PCR method, PCR clamping method, amplification refractory mutation system (ARMS), digital PCR, and competition. Competitive Allele-Specific Taqman PCR (CAST PCR), etc., all of which have their own advantages and disadvantages.

DNA sequencing is a reliable method for detecting mutations and is a more widely used method. The sequencing method has high requirements on materials and technology. The most important thing is that due to the limitation of the sequencing method itself, the sensitivity is not high, and only the mutant mutant of the target nucleic acid sequence with the content greater than 20% can be detected.

The RFLP-PCR method removes the wild-type variant similar to the mutant variant of the target nucleic acid by using a restriction enzyme before or during the PCR reaction, thereby amplifying and enriching the target nucleic acid. The purpose of the mutant variant of the fragment. There are a number of variations based on this method, including restriction enzyme cleavage site analysis PCR (RSM-PCR) methods, primer-ligated amplification (APRIL-ATM) methods at the mutation site, and the like. Although such methods have the advantages of simple design and low cost, and have good selectivity in some specific experiments, they rely on restriction endonuclease sites near the mutation site, in specific applications. The selectivity of such methods is limited.

The PCR clamping method achieves the purpose of selectively amplifying a target fragment to be detected by inhibiting amplification of a wild type variant of a target nucleic acid sequence. The use of peptide nucleic acids (PNA) or methods using locked nucleic acids (LNA) are described in the literature [Henrik et al., Nucleic Acid Research 21: 5332-5336 (1993)] and [Luo et al., Nucleic Acid Research Vol. 34, respectively. Published in No 2 e12 (2006)]. However, since the wild type variant of the target nucleic acid sequence differs from the mutant variant by only one or two or three bases, the peptide nucleic acid or the locked nucleic acid is also susceptible to binding to the mutant variant, thereby producing a false negative result.

Digital PCR (digital PCR) detects a small number of target nucleic acid sequence mutant variants by diluting the template and increasing the number of PCR reactions [Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci USA 1999; 96: 9236-41]. In theory, when the nucleus When the acid template is diluted to have only one or no nucleic acid template per PCR reaction, either no amplification is amplified in this PCR reaction, or the wild type template is amplified or the mutant template is amplified. The goal of detecting a small number of mutant variants can be achieved by combining the corresponding detection methods. In theory, this method is infinite by increasing the quantitative selectivity of the PCR reaction. However, in practice, selectivity is not only limited by the fidelity limitation of Taq DNA polymerase, but also by the number of PCRs that can be performed simultaneously. Although digital PCR-based methods have been reported to have high selectivity, as disclosed in [Bielas JH, Loeb LA. Quantification of random genomic mutations. Nat Methods 2005; 2: 285-90]. However, this method usually requires specialized instruments and chip technology. The process is very complicated and costly.

The amplification refractory mutation system (ARMS), also known as allele specific PCR (AS-PCR), is based on the principle that the Taq DNA polymerase lacks the 3'-5' exo Enzyme activity, the principle that the 3'-end base of the PCR primer must be complementary to its template DNA for efficient amplification, and appropriate primers are designed for different known mutations to detect the mutant gene. The main limiting factor of this method is that if the mutated site is a weak mismatched base sequence, the ARMS primers cannot effectively distinguish between wild-type variants and mutant variants of the target nucleic acid sequence, thereby affecting the selectivity of the method. In addition, the detection of 14 deletion mutations in common EGFR mutations requires 14 ARMS primers to achieve detection [US Premarket Approval Application number: 15047 (2016)].

Competitive Allele-Specific TaqMan PCR (CAST PCR), which blocks the amplification of wild-type variants by blocking probes, and uses ARMS primers to selectively amplify mutant variants. The combination of the two reduces the probability of erroneous binding of ARMS primers to wild-type variants to a certain extent, but this technique uses non-selective public probes and lacks efficient selective detection of mutant variants.

Summary of the invention

An object of the present invention is to provide a method for detecting a variant of a target nucleic acid sequence, which uses a closed probe having a special structure together with a selective detection probe to achieve high selectivity detection. The detection effect of the deletion mutation and the insertion mutation is more remarkable.

Another object of the present invention is to provide a detection kit for detecting a variant of a target nucleic acid sequence, which is more effective in detecting a deletion mutation or an insertion mutation.

According to a first aspect of the invention, the invention provides a composition for detecting a variant of a target nucleic acid sequence. The composition may comprise: (a) a blocking probe that specifically binds to a wild-type variant of the target nucleic acid sequence, and whose 3' end is modified with an oligonucleotide that prevents its extension; (b) detection a probe that specifically binds to a mutant variant of the target nucleic acid sequence and that produces a detection signal; (c) a primer, a primer that shares with the wild-type variant and the mutant variant of the target nucleic acid sequence.

Further, the blocked probe 3' end is prevented from extending by a non-hydroxyl group including, but not limited to, phosphorylation, amino, deoxygenation, halogenation, C3 Spacer, C6 Spacer modification. .

In the present invention, the blocking probe contains a nucleic acid double-strand stabilizing factor.

Further, the nucleic acid double-strand stabilizing factor is located outside the 5' end of the blocking probe, including but not limited to modification of a base, use of a base analog, alteration of a nucleic acid backbone, modification of a glycosyl group, preferentially Locked nucleic acids (LNA), peptide nucleic acid (PNA).

Further, the nucleic acid double-strand stabilizing factor is located at the 5' end of the blocking probe and is one or a kind of DNA minor groove binder (MGB). The combination above.

Further, in the present invention, the 5' end of the blocking probe contains a modification factor which inhibits nuclease hydrolysis.

Further, the modification factor includes a base analog, a nucleic acid backbone, a deoxyribose analog, a peptide nucleic acid or a phosphorothioate.

Further, the detection probe 5' end is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescence quenching group.

Further, the fluorescent reporter group is selected from the group consisting of FAM, TET, HEX, JOE, CY3, CY5, ROX or Texas Red; the fluorescent quenching group is selected from the group consisting of TAMRA, BHQ1, BHQ2 or CY5.

Further, the composition further comprises a polymerase, dNTPs, and/or other reagents or buffers suitable for PCR amplification.

The present invention also provides a method for detecting a variant of a target nucleic acid sequence, which mixes a sample of the nucleic acid to be tested with a composition of the above-mentioned detection target nucleic acid sequence variant, and performs an amplification reaction by detecting a change in the detection signal of the detection probe. The amplicon is detected to detect a mutant variant of the target nucleic acid in the nucleic acid sample to be tested.

The present invention further includes quantifying the mutant variant by detecting a change in the detection signal of the probe.

Further, the amplification reaction includes, but is not limited to, isothermal amplification technology, polymerase chain reaction (PCR), including but not limited to loop-mediated isothermal amplification (LAMP), and dependence. Nucleic acid sequence amplification technology (NASBA), rolling circle amplification technology (RCA), single primer isothermal amplification (SPIA), helicase-dependent isothermal amplification (HAD), strand-substitution amplification ( SDA), rapid isothermal detection amplification technique (RIDA), nicking endonuclease nucleic acid constant temperature amplification technique (NEMA).

In the present invention, the amplification reaction is real-time fluorescent quantitative PCR.

Further, the mutant variant is a point mutation, an insertion mutation, and a deletion mutation.

According to a second aspect of the present invention, the present invention provides a detection kit for detecting a variant of a target nucleic acid sequence, the kit comprising the following components: a blocking probe, a detection probe and a primer;

The blocking probe specifically binds to a wild type variant of the target nucleic acid sequence, and the 3' end thereof is modified with an oligonucleotide that prevents its extension;

The detection probe specifically binds to a mutant variant of the target nucleic acid sequence and is capable of generating a detection signal;

The primer is a common primer for a wild type variant and a mutant variant of the target nucleic acid sequence.

Further, the blocked probe 3' end is subjected to non-hydroxyl group modification, including but not limited to phosphorylation, amino group, deoxygenation, halogenation, C3 Spacer, C6 Spacer modification.

In the present invention, the blocking probe contains a nucleic acid double-strand stabilizing factor.

Further, the nucleic acid double-strand stabilizing factor is located outside the 5' end of the blocking probe, including but not limited to modification of a base, use of a base analog, alteration of a nucleic acid backbone, modification of a glycosyl group, preferentially Locked nucleic acids (LNA), peptide nucleic acid (PNA).

Further, the nucleic acid double-strand stabilizing factor is located at the 5' end of the blocking probe and is a combination of one or more of DNA minor groove binders (MGBs).

Further, the 5' end of the blocking probe contains a nuclease inhibiting hydrolysis Decorative factor.

Further, the modification factor includes a base analog, a nucleic acid backbone, a deoxyribose analog, a peptide nucleic acid or a phosphorothioate.

Further, the detection probe 5' end is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescence quenching group.

Further, the fluorescent reporter group is selected from the group consisting of FAM, TET, HEX, JOE, CY3, CY5, ROX or Texas Red; the fluorescent quenching group is selected from the group consisting of TAMRA, BHQ1, BHQ2 or CY5.

Further, the reaction mixture further comprises a polymerase, dNTPs, and/or other reagents or buffers suitable for PCR amplification.

The present invention has the following positive effects:

1. Excellent performance: The present invention combines selective amplification and selective detection. The blocking probe preferentially binds to the wild type variant, and the selective detection probe preferentially binds to the mutant variant, that is, the binding probe is used. The wild type variant of the target nucleic acid sequence is such that the wild type variant cannot be efficiently amplified in the amplification reaction, thereby selectively amplifying the mutant variant of the target nucleic acid sequence to form a mutant variant amplicon. At the same time, the selective detection probe binds to the mutant variant, and the mutant variant amplicator is detected by detecting the change of the probe fluorescence signal, so that even if the wild type variant is not bound by the blocking probe, the wild type variant is formed. The amplicon, because it cannot bind to the selective detection probe, can not produce a detection signal, thereby preventing the blocking probe from binding to the mutant variant, and releasing the blocking probe to inhibit the mutant variant, thereby avoiding false negative results. . Therefore, the method can detect trace mutant variants in a large number of wild type variants. In the traditional allele-specific PCR (AS-PCR) method, primers using 3'-end mismatches are equivalent to artificially introduced mutant variants when erroneously extended, and such artificially introduced mutant variants are In each subsequent cycle, it is amplified as a mutant variant, but the present invention does not have this problem. In the present invention, even if the wild type variant is amplified in the PCR round reaction, amplification is performed. The product is still easily bound by Blocker in the next round of reactions and cannot be efficiently amplified, thereby effectively inhibiting the amplification efficiency of the wild type variant, and the selective detection probe is used for selective detection.

2. The system is simple and it is not easy to have side reactions: Since the invention adopts common primers, only one pair of primers can simultaneously detect multiple deletions and/or insertion mutations, and the formation of dimers between multiple primers can be effectively avoided. For example, when using the ARMS PCR method to detect 14 deletion mutations of a common EGFR mutation, 14 ARMS primers are required to achieve detection [US Premarket Approval Application number: 15047 (2016)], and only one of the techniques of the present invention is required. The primer can realize the detection of all deletion mutations, effectively avoiding the formation of dimers between multiple primers, thereby reducing the occurrence of side reactions, improving the efficiency of amplification detection, and enhancing the detection sensitivity.

3. Wide application, the invention can be widely applied in the fields of nucleic acid amplification, tumor in vitro diagnosis, genotyping and the like.

DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below. Obviously, the drawings in the following description are only It is a certain embodiment of the present invention, and other drawings can be obtained from those skilled in the art without any creative work.

Figure 1 is an amplification diagram of the mutant sequence (EGFR2235-2249del15) and the wild type sequence in the case of addition of a selective detection probe;

Figure 2 Mutant sequence (EGFR2235-2249del15), wild type sequence and two sequences Amplification pattern of the mixed sequence in the case of adding a blocking probe and a selective detection probe;

Figure 3 is an amplification diagram of the mutant sequence (EGFR2236-2253del18) and the wild type sequence in the case of addition of a selective detection probe;

Figure 4 is an amplification diagram of the mutant sequence (EGFR2236-2253del18), the wild type sequence, and the mixed sequence of the two sequences in the case of adding a blocking probe and a selective detection probe;

Figure 5 is an amplification diagram of the wild-type sequence corresponding to the mutant sequence (EGFR 2319-2320insCAC) in the presence of a occluded probe and without a occlusion probe;

Figure 6 is an amplification diagram of the mutant sequence (EGFR 2319-2320insCAC), the wild type sequence, and the mixed sequence of the two sequences in the case of adding a blocking probe and a selective detection probe;

Figure 7 is an amplification map of the wild-type sequence corresponding to the mutant sequence (EGFR2310-2311 insGGT) in the presence of a occluded probe and without a occlusion probe;

Figure 8. Amplification sequence of the mutant sequence (EGFR2310-2311 insGGT), the wild type sequence and the mixed sequence of the two sequences in the case of the addition of a blocking probe and a selective detection probe.

detailed description

In order to detect a small amount of a nucleic acid variant to be detected which is present in a large number of non-detection nucleic acid variants, the inventors conducted a lot of research work and proposed the technical scheme of the present invention. Those skilled in the art can learn from the contents of this document and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The application of the present invention has been described in terms of the preferred embodiments thereof, and it is obvious that those skilled in the art can make and modify and combine the application described herein to implement and apply the present technology without departing from the scope of the present invention. .

In one aspect, the invention provides a composition for detecting a variant of a target nucleic acid sequence. The composition may comprise: (a) a blocking probe that specifically binds to a wild-type variant of the target nucleic acid sequence, and whose 3' end is modified with an oligonucleotide that prevents its extension; (b) The detection probe specifically binds to the mutant variant of the target nucleic acid sequence and is capable of generating a detection signal; (c) a primer, a primer shared with the wild type variant and the mutant variant of the target nucleic acid sequence.

In some embodiments, the 3' end of the blocking probe is prevented from extending by non-hydroxyl groups including, but not limited to, phosphorylation, amino, deoxygenation, halogenation, C3 Spacer, C6 Spacer Modification.

In some embodiments, the blocking probe comprises a nucleic acid double strand stabilizing factor.

In some embodiments, the nucleic acid double-strand stabilizing factor is located outside of the 5' end of the blocking probe, including but not limited to, modification of the base, use of a base analog, alteration of the nucleic acid backbone, modification of a glycosyl group.

In some preferred embodiments, the nucleic acid double-strand stabilizing factor is locked nucleic acid (LNA), peptide nucleic acid (PNA).

In some embodiments, the nucleic acid double-strand stabilizing factor is located at the 5' end of the blocking probe and is a combination of one or more of a DNA minor groove binder (MGB).

In some embodiments, the 5' end of the blocking probe contains a modification factor that inhibits nuclease hydrolysis.

Wherein the modifying factor comprises a base analog, a nucleic acid backbone (peptide nucleic acid or phosphorothioate), a deoxyribose analog, a peptide nucleic acid or a phosphorothioate.

In some embodiments, the composition further comprises a polymerase, dNTPs, and/or other reagents or buffers suitable for PCR amplification.

Further, the detection probe 5' end is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescence quenching group. For the fluorescence quantitative detection method, when the probe is intact, 5' The fluorescent reporter group at the end is restricted by the 3'-end fluorescence quenching group and does not emit fluorescence. When the detection probe binds to the mutant variant of the target nucleic acid sequence, when PCR is amplified, the 3'-5' exonuclease activity of the Taq enzyme degrades the probe, allowing the reporter fluorophore and quenching fluorescence. The group is separated, and the fluorescent reporter group at the 5' end is released, emits fluorescence, and the purpose of detecting fluorescence is achieved by real-time PCR.

Further, the fluorescent reporter group is selected from the group consisting of FAM, TET, HEX, JOE, CY3, CY5, ROX or Texas Red; the fluorescent quenching group is selected from the group consisting of TAMRA, BHQ1, BHQ2 or CY5.

In another aspect, the present invention also provides a method for detecting a variant of a target nucleic acid sequence, mixing a sample of the nucleic acid to be tested with a composition of the above-mentioned detection target nucleic acid sequence variant, and performing an amplification reaction by detecting a detection signal of the detection probe. The mutation is detected to detect a mutant variant of the target nucleic acid in the nucleic acid sample to be tested.

Further, the mutant variant can also be quantified by detecting a change in the detection signal of the probe.

In some embodiments, the amplification reaction includes, but is not limited to, isothermal amplification techniques, polymerase chain reaction (PCR).

Further, the isothermal amplification techniques include, but are not limited to, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-dependent amplification (NASBA), rolling circle amplification (RCA), single primer isothermal amplification. Technology (SPIA), helicase-dependent isothermal amplification (HAD), strand-substitution amplification (SDA), rapid isothermal amplification (RIDA), nicking enzyme nucleic acid constant amplification (NEMA) .

In some preferred embodiments, the amplification reaction is real-time fluorescent quantitative PCR.

In some embodiments, the mutant variant is a point mutation, an insertion mutation, a deletion mutation.

According to a second aspect of the present invention, the present invention provides a detection kit for detecting a variant of a target nucleic acid sequence, the kit comprising the following components: a blocking probe, a detection probe and a primer;

The blocking probe specifically binds to a wild type variant of the target nucleic acid sequence, and the 3' end thereof is modified with an oligonucleotide that prevents its extension;

The detection probe specifically binds to a mutant variant of the target nucleic acid sequence and is capable of generating a detection signal;

The primer is a common primer for a wild type variant and a mutant variant of the target nucleic acid sequence.

In some embodiments, the 3' end of the blocking probe is prevented from extending by non-hydroxyl groups including, but not limited to, phosphorylation, amino, deoxygenation, halogenation, C3 Spacer, C6 Spacer Modification.

In some embodiments, the blocking probe comprises a nucleic acid double strand stabilizing factor.

In some embodiments, the nucleic acid double-strand stabilizing factor is located outside of the 5' end of the blocking probe, including but not limited to, modification of the base, use of a base analog, alteration of the nucleic acid backbone, modification of a glycosyl group.

In some preferred embodiments, the nucleic acid double-strand stabilizing factor is locked nucleic acid (LNA), peptide nucleic acid (PNA).

In some embodiments, the nucleic acid double-strand stabilizing factor is located at the 5' end of the blocking probe and is a combination of one or more of a DNA minor groove binder (MGB).

In some embodiments, the 5' end of the blocking probe contains a modification factor that inhibits nuclease hydrolysis.

Wherein the modification factor comprises a base analog, a nucleic acid backbone (peptide nucleic acid or thiophosphorus) An acid ester), a deoxyribose analog, a peptide nucleic acid or a phosphorothioate.

In some embodiments, the 5' end of the detection probe is labeled with a fluorescent reporter group and the 3' end is labeled with a fluorescence quencher group.

Further, the fluorescent reporter group is selected from the group consisting of FAM, TET, HEX, JOE, CY3, CY5, ROX or Texas Red; the fluorescent quenching group is selected from the group consisting of TAMRA, BHQ1, BHQ2 or CY5.

In some embodiments, the composition further comprises a polymerase, dNTPs, and/or other reagents or buffers suitable for PCR amplification. Such as glycerin, Tris·HCl, KCl, MgCl ₂ and the like.

Unless otherwise defined, all scientific or technical terms of the patent are in accordance with the ordinary understanding of the ordinary person in the art. The general definition of most of the terminology in the literature in the following literature: [Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994)]; [The Cambridge Dictionary of Science and Technology (Walker ed., 1988) [The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991)]; [Hale & Marham, The Harper Collins Dictionary of Biology (1991)] Unless otherwise defined, in this patent The terminology used is consistent with the description of the term in the above documents.

The term "nucleotide" generally refers to a compound formed by the attachment of an nucleoside to an acidic molecule or group, for example, a phosphate of a nucleoside, usually having one, two or three phosphate groups. The valence is attached to the 5 position of the sugar group of the nucleoside. In some cases, the definition of a nucleotide also includes homologs or analogs of some typical nucleotides.

The term "oligonucleotide" refers to a polymer formed by the covalent bond between nucleotides. An oligonucleotide typically comprises at least 3 nucleotides. In some cases, oligonucleotides may also contain phosphorus ammonia [Beaucage et al. (1993) Tetrahedron 49(10): 1925], phosphorothioate [Mag et al. (1991) Nucleic Acids Res. 19:1437; and US Pat. No.5644048)], dithiophosphate [Briu et al. (1989) J. Am. Chem. Soc. 111:2321], O-methylphosphorus linkage [ Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press (1992))], peptide nucleic acid backbone linkage [Egholm (1992) J. Am. Chem. Soc. 114: 1895]. Other oligonucleotides also include those that are positively charged [Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6097], non-ionic scaffold (US Pat. Nos. 5,386,023, 5,637,684) , 5602240, 5216141 and 4469863) and non-ribose backbones (US Pat. Nos. 5235033 and 5034506). Oligonucleotides containing one or more carbocyclic sugars are also in the definition of nucleic acids [Jenkins et al. (1995) Chem. Soc. Rev. pp. 169-176]. The modification of the ribose-phosphate backbone for the purpose of improving the stability of the molecule under specific conditions or for labeling oligonucleotides is also included within the definition of the oligonucleotide. Oligonucleotides may also include various spacer modifications, such as C3 Spacer, C9 Spacer, C18 Spacer, and the like.

The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA-RNA hybrids, oligonucleotides, aptamers, peptide nucleic acids (PNAs), PNA-DNA hybrids, PNA. - RNA hybrids and the like. Includes all covalently linked nucleotides in linear form (single or double stranded) or branched form. A typical nucleic acid is usually single-stranded or double-stranded and contains a diphosphate bond.

The term "target nucleic acid sequence" refers to a nucleic acid sequence to be amplified which serves as a template for nucleic acid amplification.

The terms "nucleic acid sequence variant" and "nucleic acid variant" refer to a specific nucleic acid sequence, and there are differences between different variants. This difference may be a single or multiple bases, or may be an insertion or a deletion. , translocation or a combination of all of the above.

The term "wild-type variant" refers to a naturally occurring "normal" gene sequence that appears most frequently in the same gene sequence in most populations.

The term "mutant variant" refers to a nucleic acid sequence that differs from a "wild-type variant". This difference may be a single or multiple bases, or an insertion or deletion or a combination of all of the above, for example, a mutated nucleic acid sequence in a tumor cell. In some embodiments, the proportion of mutant variants is small compared to wild-type variants.

The term "amplification" refers to the process of increasing the number of nucleic acid fragments of interest under the action of a nucleic acid polymerase, including but not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR), and nucleic acid sequence extension. Increase (NASBA), transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), and the like.

In an embodiment of the invention, amplification refers to polymerase chain reaction (PCR). The template is denatured and melted, and the oligonucleotide primer hybridizes with the template annealing, and with the extension of the nucleotide addition, the cycle is repeated for a certain number of rounds to achieve an increase in the nucleotide fragment of interest.

The term "mutation" refers to a change in the genetics of a cell. It includes point mutations caused by single base changes, or deletions, duplications and insertions of multiple bases.

The "nucleic acid double-strand stabilizing factor" in this patent refers to a compound or group that can help stabilize the double-stranded structure of a nucleic acid, which may be a locked nucleic acid, or a peptide nucleic acid or a DNA minor groove conjugate. Preferably, the modification is a DNA minor groove conjugate. It has previously been reported in the literature that DNA minor groove conjugate molecules can significantly increase the annealing temperature of hybrid nucleic acid dimers, usually the DNA minor groove conjugate is attached to the 3' end of the oligonucleotide fragment. Since the DNA zoning conjugate can significantly increase the annealing temperature of the hybrid nucleic acid dimer, different nucleic acid variants have different degrees of complementarity with the modified oligonucleotide fragments, resulting in a larger difference in annealing temperature, thereby producing a difference. The effect of blocking the extension of the nucleic acid polymerase. In the present invention, preferably, the DNA minor groove conjugate is ligated to the 5' end of the oligonucleotide.

The term "small groove conjugate" refers to a compound that binds to the minor groove of DNA. The surface of the DNA double helix has two grooves, named big groove and small groove. DNA ditch phase There are more hydrogen bond donors, receptors, charges and other information than DNA ditch, and intracellular proteins prefer to recognize sites in the ditch to regulate gene expression or perform other intracellular functions.

DNA sulcus is a target for some antibiotics in nature, such as netropsin, distamycin. The dissociation constants of these two compounds bound to DNA are all on the order of 10 to 5 M and exhibit a preference for the DNA AT-rich region. These two compounds have certain limitations in terms of selectivity, toxicity, and the like. Researchers have found many similar compounds to overcome these limitations, such as the compounds mentioned in the following review [Sondhi et al. (Curr. Med. Chem. 4, 313 (1997)), [Reddy et al. (Pharmacology & Therapeutics 84 , 1 (1999)], Wemmer (Biopolymers 52, 197 (2001)], [Dervan (Bioorg. Med. Chem. 9, 2215 (2001)].

Still others are said to prefer to bind DNA GC base pair regions, such as the compounds mentioned in the following literature [Anti-Cancer Drug Design 5, 3 (1990)], [Proc. Natl. Acad. Sci. USA 89, 7586 (1992)], [Biochemistry 32, 4237 (1993)], [Science 266, 647 (1994)], [Anti-Cancer Drug Design 10, 155 (1995)], [Bioorg Med. Chem. 8, 985 (2000)], [Mol .Biol. 34, 357 (2000)]. Different spindles (netropsin), analogs of distamycin have been discovered, for example [J Am. Chem. Soc. 114 (15), 5591 (1992)], [Biochemistry 31, 8349 (1992)] , [Bioconjugate Chem. 5, 475 (1994)], [Biochem. Biophys. Res. Commun. 222, 764 (1996)], [J Med. Chem. 43, 3257 (2000)], [Tetrahedron 56, 5225 (2000)], [Molecular Pharmacology 54, 280 (1998)], [Bioorg Med. Chem. Lett. 6 (18), 2169 (1996)], [J. Med. Chem. 45, 805 (2002)], [Bioorg. Med. Chem. Lett. 12,2007 (2002)], (international patent applications WO 97/28123, WO 98/21202, WO 01/74898 and WO 02/00650, US patent numbers 4,912, 199, 5, 273, 991, 5, 637, 621, 5, 698, 674 and 5, 753, 629). Netropsin, a polypeptide homolog of distamycin, is disclosed in the following patent WO/2003/059881.

The term "sequencing method" refers to the analysis of the base sequence of a specific nucleic acid fragment, that is, the arrangement of adenine (A), thymine (T), cytosine (C), and guanine (G). The Sanger method starts at a fixed point based on nucleotides, randomly terminates at a specific base, and is fluorescently labeled after each base to produce adenine (A) and thymine (T). Four sets of nucleotides of different lengths, cytosine (C) and guanine (G), were detected by electrophoresis on a urea-denatured PAGE gel to obtain a visible DNA base sequence. In circular array synthesis sequencing, sequences are indirectly determined by reading optical signals released by DNA polymerase or DNA ligase to link bases to DNA strands, with the aid of fluorescent or chemiluminescent substances. . The novel nanopore sequencing method uses electrophoresis technology to drive individual molecules through the nanopore to perform sequencing by electrophoresis. Since the diameter of the nanopore is very small, only a single nucleic acid polymer is allowed to pass, and different single bases have different charging properties, and the base class passed can be detected by the difference of electrical signals, thereby realizing sequencing. There are many methods for nucleic acid sequencing, and new methods are constantly being developed.

In an embodiment of the invention, the nucleic acid amplification assay is a fluorescent quantitative PCR assay. In a real-time PCR reaction, the parameter that measures amplification is the Ct value, and the earlier Ct value reflects that the signal reaches the threshold faster. The difference in Ct between different nucleic acid variants in the amplified sample often reflects the difference in amplification efficiency of the amplification system for different nucleic acid variants, and further reflects the selectivity of the amplification system.

Detection of amplification products There are many methods in the art. These methods include the use of fluorescently labeled probes or various dyes that bind to nucleic acids. These tests may be one or more of the specific detection of nucleic acid variants, or non-selective detection of all Nucleic acid signal. Detection of the amplified product may occur after completion of the amplification reaction, such as by gel electrophoresis, or by staining the nucleic acid. In addition, detection of amplification products can also occur during the course of the amplification reaction.

Example 1: Selective Amplification and Detection of Deletion Mutations Using the Methods of the Invention

In this embodiment, the amount of two kinds of the target nucleic acid sequence variants were added to 100 copies and 10 ⁶ copies, a variant thereof is inserted into a plasmid DNA EGFR 2235-2249del15 mutant sequence, while the other variants are The same plasmid was inserted but the EGFR wild type sequence was inserted.

The EGFR mutation sequence is:

The wild type sequence of EGFR is:

The forward primer sequence is: DF-GACTCTGGATCCCAGAAGGTGA

The reverse primer sequence is: DR-GGGCCTGAGGTTCAGAGCC

The blocking probe sequence is: DB-GGAATTAAGAGAAGCAACATCTCCGAAA (5' end MGB modification, 3' end C3 Spacer)

The detection probe sequence is: DPF1-TCGCTATCAAAACATCT (5'-end FAM, 3'-end MGB)

The PCR reaction system is 25μl, each reaction system contains 2% glycerol, dATP, dCTP, dGTP, dTTP each 200μM, forward primer, reverse primer each 200nM, detection probe 200nM, closed probe 500nM and 1 unit Hot start Taq DNA polymerase. One of the units refers to the amount of enzyme required to incorporate 10 nmol of dNTPs at 72 ° C for 30 minutes.

PCR reactions and subsequent data analysis were performed using a Roche LightCycler 480 fluorescence quantitative PCR machine. The PCR thermal cycling conditions were 95 ° C, 10 min; 50 cycles (95 ° C, 15 s; 60 ° C, 40 s, single cycle); 37 ° C, 30 s.

The experimental results are shown in Figures 1 and 2, and the experimental results are reflected by the fluorescence change at 465 nM-510 nM.

In Figure 1, it can be seen that the 10 ⁶ copies wild type template has very low amplification efficiency after adding the detection probe, and has substantially no amplification, thereby indicating that the detection probe has extremely high detection selectivity.

In Figure 2, it can be seen that the 10 ⁶ copies wild type template has no amplification at all after the addition of the blocking probe and the detection probe. Closing the mutant-type template hybrid template (100 copies of mutant and wild-type 10 ⁶ copies) were obtained when the probe and detection probe coexist efficient amplification, the presence of the blocker probe does not interfere with the detection of the mutant-type template, while the detection The presence of the probe also does not interfere with the binding of the blocked probe to the wild type template.

Example 2: Selective amplification and detection of deletion mutations using the methods described herein

In this embodiment, the amount of two kinds of the target nucleic acid sequence variants were added to 100 copies and 10 ⁶ copies, a variant thereof is inserted into a plasmid DNA EGFR 2236-2253del18 mutant sequence, while the other variants are The same plasmid was inserted but the EGFR wild type sequence was inserted.

The EGFR mutation sequence is:

The wild type sequence of EGFR is:

The forward primer sequence is: DF-GACTCTGGATCCCAGAAGGTGA

The reverse primer sequence is: DR-GGGCCTGAGGTTCAGAGCC

The blocking probe sequence is: DB-GGAATTAAGAGAAGCAACATCTCCGAAA (5' end MGB modification, 3' end C3Spacer)

The detection probe sequence is: DPF2-TCGCTATCAAGTCTCCGAAAGCCAACA (5'-end FAM, 3'-end BHQ1)

The PCR reaction system is 25μl, each reaction system contains 2% glycerol, dATP, dCTP, dGTP, dTTP each 200μM, forward primer, reverse primer each 200nM, detection probe 200nM, closed probe 500nM and 1 unit Hot start Taq DNA polymerase.

PCR reactions and subsequent data analysis were performed using a Roche LightCycler 480 fluorescence quantitative PCR machine. The PCR thermal cycling conditions were 95 ° C, 10 min; 50 cycles (95 ° C, 15 s; 60 ° C, 40 s, single cycle); 37 ° C, 30 s.

The experimental results are shown in Figures 3 and 4, and the experimental results are reflected by the fluorescence change at 465 nM-510 nM.

In Figure 3, it can be seen that the 10 ⁶ copies wild type template has very low amplification efficiency after adding the detection probe, and has substantially no amplification, thereby indicating that the detection probe has extremely high detection selectivity.

In Figure 4, it can be seen that the 10 ⁶ copies wild type template has no amplification at all after the addition of the blocking probe and the detection probe. When the blocking probe and the detection probe coexist, the mutant template and the mixed template (100copie mutant and 106 ⁶ wild type) are effectively amplified, and the presence of the blocking probe does not interfere with the detection of the mutant template, and detection The presence of the needle also does not interfere with the binding of the blocking probe to the wild type template.

Example 3: Selective amplification and detection of insertional mutations using the methods described herein

In this embodiment, the amount of two kinds of the target nucleic acid sequence variants were added to 100 copies and 10 ⁶ copies, a variant thereof is inserted into a plasmid DNA EGFR 2319-2320insCAC mutant sequence, while the other variants are The same plasmid was inserted but the EGFR wild type sequence was inserted.

The EGFR mutation sequence is:

The wild type sequence of EGFR is:

The forward primer sequence is: INF-CTACGTGATGGCCAGCGTG

The reverse primer sequence is: INR-CATTCATGCGTCTTCACCTGG

The blocking probe sequence is: INB-ACAACCCCCACGTGT (5' end MGB modification, 3' end C3 Spacer)

The detection probe sequence is: INPH1-CCCCACCACGTGTGC (5' end HEX, 3' end MGB)

The PCR reaction system is 25μl, each reaction system contains 2% glycerol, dATP, dCTP, dGTP, dTTP each 200μM, forward primer, reverse primer each 200nM, detection probe 200nM, closed probe 500nM and 1 unit Hot start Taq DNA polymerase.

PCR reactions and subsequent data analysis were performed using a Roche LightCycler 480 fluorescence quantitative PCR machine. The PCR thermal cycling conditions were 95 ° C, 10 min; 50 cycles (95 ° C, 15 s; 60 ° C, 40 s, cycle); 37 ° C, 30 s.

The experimental results are shown in Figures 5 and 6, and the experimental results are reflected by the fluorescence change at 533 nM-580 nM.

It can be seen in Figure 5 that the 10 ⁶ copies wild-type template was amplified significantly when there was no blocking probe, and there was substantially no amplification after the blocking probe was added.

In Figure 6 the closure is visible in the coexistence of a probe similar to the probe and detecting hybrid template amplification effect mutant templates (100copies 10 ⁶ copies of mutant and wild type) to amplify the effect, the presence of the probe does not interfere with the closure Detection of the mutant template, while detecting the presence of the probe, does not interfere with the binding of the blocking probe to the wild-type template.

The above experimental results demonstrate that selective amplification and selective detection of insertional mutations are achieved using the methods described herein.

Example 4: Selective Amplification and Detection of Insertion Mutations Using the Methods of the Invention

In this embodiment, the amount of two kinds of the target nucleic acid sequence variants were added to 100 copies and 10 ⁶ copies, a variant thereof is inserted into a plasmid DNA sequence EGFR 2310-2311insGGT, while the other variants are identical The plasmid was inserted into the wild type sequence of EGFR.

The EGFR mutation sequence is:

The wild type sequence of EGFR is:

The forward primer sequence is: INF-CTACGTGATGGCCAGCGTG

The reverse primer sequence is: INR-CATTCATGCGTCTTCACCTGG

The blocking probe sequence is: INB-ACAACCCCCACGTGT (5' end MGB modification, 3' end C3 Spacer)

The detection probe sequence is: INPH2-GTGGACGGTAACCC (5' end HEX, 3' end MGB)

The PCR reaction system is 25μl, each reaction system contains 2% glycerol, dATP, dCTP, dGTP, dTTP each 200μM, forward primer, reverse primer each 200nM, check The probe was 200 nM, the probe was blocked at 500 nM and 1 unit of hot-start Taq DNA polymerase.

PCR reactions and subsequent data analysis were performed using a Roche LightCycler 480 fluorescence quantitative PCR machine. The PCR thermal cycling conditions were 95 ° C, 10 min; 50 cycles (95 ° C, 15 s; 60 ° C, 40 s, single cycle); 37 ° C, 30 s.

The experimental results are shown in Figures 7 and 8, and the experimental results are reflected by the fluorescence change at 533 nM-580 nM.

It can be seen in Figure 7 that the 10 ^{^6} wild-type template was amplified significantly when there was no blocking probe, and there was substantially no amplification after the blocking probe was added.

It can be seen in Figure 8 that the amplification effect of the wild-type template when the blocking probe and the detection probe coexist is similar to that of the mixed template (100copies mutant and 106 ⁶ wild type), and the presence of the blocked probe does not interfere. Detection of the mutant template, while detecting the presence of the probe, does not interfere with the binding of the blocking probe to the wild-type template.

The above experimental results demonstrate that selective amplification and selective detection of insertional mutations are achieved using the methods provided herein.

The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments are obvious to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein, but the scope of the invention.

Claims (27)

A composition for detecting a variant of a target nucleic acid sequence, comprising: a blocking probe, a detection probe, and a primer; The blocking probe specifically binds to a wild type variant of the target nucleic acid sequence, and the 3' end thereof is modified with an oligonucleotide that prevents its extension; The detection probe specifically binds to a mutant variant of the target nucleic acid sequence and is capable of generating a detection signal; The primer is a common primer for a wild type variant and a mutant variant of the target nucleic acid sequence. The composition of claim 1 wherein the modification of the 3' end of the blocking probe is selected from the group consisting of phosphorylation, amino, deoxygenation, halogenation, C3 Spacer, C6 Spacer. The composition of claim 1 further comprising a nucleic acid double strand stabilizing factor. The composition according to claim 3, wherein the nucleic acid double-strand stabilizing factor is located at a position other than the 5' end of the blocking probe, and is selected from the group consisting of a modification of a base, use of a base analog, alteration of a nucleic acid skeleton, and modification of a glycosyl group. . The composition according to claim 4, wherein the nucleic acid double-strand stabilizing factor is a locked nucleic acid or a peptide nucleic acid. The composition according to claim 3, wherein the nucleic acid double-strand stabilizing factor is located at the 5' end of the blocking probe and is one or a combination of one or more of the DNA minor groove conjugates/analogs. The composition according to claim 3, wherein the blocking probe 5' end contains a modification factor which inhibits nuclease hydrolysis. The composition according to claim 7, wherein the modification factor is selected from the group consisting of a base analog, a nucleic acid backbone, and a deoxyribose analog. The composition according to claim 1, wherein the detection probe 5' is labeled with a fluorescent reporter group at its end, and the 3' end is labeled with a fluorescence quenching group. The composition of claim 9 wherein said fluorescent reporter group is selected from the group consisting of FAM, TET, HEX, JOE, CY3, CY5, ROX or Texas Red; the fluorescent quenching group is selected from the group consisting of TAMRA, BHQ1, BHQ2 or CY5. The composition of claim 1 further comprising a polymerase, dNTPs, and/or other reagents or buffers suitable for PCR amplification. A method for detecting a variant of a target nucleic acid sequence, the nucleic acid sample to be tested is mixed with the composition according to any one of claims 1 to 11, and the amplification reaction is detected by detecting a change in the detection signal of the detection probe. An amplicon to thereby detect a mutant variant of the target nucleic acid in the nucleic acid sample to be tested. The method of claim 12, wherein the amplification reaction is selected from the group consisting of isothermal amplification techniques, polymerase chain reaction (PCR). The method according to claim 13, wherein the isothermal amplification technique is selected from the group consisting of a loop-mediated isothermal amplification technique (LAMP), a nucleic acid sequence-dependent amplification technique (NASBA), a rolling circle amplification technique (RCA), and a single Primer Isothermal Amplification (SPIA), helicase-dependent isothermal amplification (HAD), strand-substitution amplification (SDA), rapid isothermal detection amplification (RIDA), cleavage of endonuclease nucleic acid Technology (NEMA). The method of claim 12, wherein the amplification reaction is real-time fluorescent quantitative PCR. The method according to claim 12, wherein the mutant variant is a point mutation, an insertion mutation or a deletion mutation. A kit for detecting a variant of a target nucleic acid sequence, comprising the following components: a blocking probe, a detection probe, and a primer; The blocking probe specifically binds to a wild type variant of the target nucleic acid sequence, and the 3' end thereof is modified with an oligonucleotide that prevents its extension; The detection probe specifically binds to a mutant variant of the target nucleic acid sequence and is capable of generating a detection signal; The primer is a common primer for a wild type variant and a mutant variant of the target nucleic acid sequence. The kit according to claim 17, wherein the closure of the 3' end of the closure probe The decoration is selected from the group consisting of phosphorylation, amino group, deoxygenation, halogenation, C3 Spacer, and C6 Spacer. The kit according to claim 17, wherein the blocking probe further comprises a nucleic acid double-strand stabilizing factor. The kit according to claim 19, wherein the nucleic acid double-strand stabilizing factor is located at a position other than the 5' end of the blocking probe, and is selected from the group consisting of a modification of a base, use of a base analog, alteration of a nucleic acid skeleton, and modification of a glycosyl group. . The kit according to claim 20, wherein the nucleic acid double-strand stabilizing factor is a locked nucleic acid or a peptide nucleic acid. The kit according to claim 21, wherein said nucleic acid double-strand stabilizing factor is located at the 5' end of the blocking probe and is one or a combination of one or more of DNA minor groove binders/analogs. The kit according to claim 17, wherein the blocking probe 5' end contains a modification factor which inhibits nuclease hydrolysis. The kit according to claim 23, wherein the modification factor is selected from the group consisting of a base analog, a nucleic acid backbone, and a deoxyribose analog. The kit according to claim 17, wherein the detection probe 5' is end-labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescence quenching group. The kit according to claim 25, wherein the fluorescent reporter group is selected from the group consisting of FAM, TET, HEX, JOE, CY3, CY5, ROX or Texas Red; and the fluorescence quenching group is selected from the group consisting of TAMRA, BHQ1, BHQ2 or CY5. The kit of claim 17 further comprising a polymerase, dNTPs, and/or other reagents or buffers suitable for PCR amplification.

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