WO2006070667A1

WO2006070667A1 - Method of detecting mutation in egfr gene and detection kit

Info

Publication number: WO2006070667A1
Application number: PCT/JP2005/023486
Authority: WO
Inventors: Masamitsu Shimada; Fumitsugu Hino; Ikunoshin Kato
Original assignee: Takara Bio Inc.
Priority date: 2004-12-28
Filing date: 2005-12-21
Publication date: 2006-07-06
Also published as: JPWO2006070667A1

Abstract

A method whereby a deficient mutation in EGFR gene, which relates to the estimation of a disease and a drug effect and to a side effect, is quickly, conveniently and highly sensitively detected; and a kit therefor.

Description

Specification

EGFR gene mutation detection method and detection kit

Technical field

[0001] The present invention relates to a method and kit for rapidly, simply, and sensitively detecting EGFR gene deletion mutations related to disease prediction, drug efficacy, and side effects.

Background art

In recent years, it has been revealed that various mutations (point mutations, deletions, insertions, duplications) at the gene level are closely related not only to genetic diseases but also to lifestyle-related diseases. Furthermore, pharmacogenomic analysis such as drug efficacy prediction and side effect prediction is being applied to clinical practice. In particular, the relationship between an EGFR inhibitor, which is a therapeutic agent for lung cancer, and EGFR mutation has been clarified (see Non-Patent Documents 1, 2, 3, and 4).

As a method for detecting a point mutation among the above gene mutations, various methods have been reported (see Non-Patent Document 5). For example, a method for examining the presence or absence of nucleic acid amplification using a primer having a mutation-specific sequence at the 3 ′ end (see Patent Document 1), a method using probe technology (see TaqMan method, Patent Documents 2 and 3), Invader Method (see patent document 4), Cycling probe technology (see non-patent document 6), CataCleave probe method (see non-patent document 7), Cycleave method (see patent document 5), Hybridization probe method (see patent document 6) , Scorpion method (see Non-Patent Document 8), sequencing method, single-base extension method, etc. These methods are widely used for Ras mutation and various SNP typing, for example, and can be applied to the analysis of point mutations in EGFR (for example, L-> R mutation at codon 858 in exon 21).

[0003] Also, deletion mutation analysis has been carried out by sequencing and high performance electrophoresis.

For example, in the example represented by the + / Δ32 deletion mutation of the CC chemokine receptor 5 (CCR5) gene, since the deletion pattern of the gene deletion mutation is constant, nucleic acid amplification with normal primers for the normal sequence Then, using two types of probes for normal sequence and defective junction region, such as TaqMan, the presence and intensity of the signal are examined and typed (Non-patent Document 9). [0004] Patent Document 1: Japanese Patent No. 2853864

Patent Document 2: US Pat. No. 5, 210,015 specification

Patent Document 3: US Pat. No. 5,487,972

Patent Document 4: US Pat. No. 5,846,717

Patent Document 5: Pamphlet of International Publication No. 03Z074696

Patent Document 6: Pamphlet of International Publication No. 97Z46714

Non-patent literature l: Science, 304, p. 1497-1500 (2004)

Non-Patent Document 2: New England Journal of Mededicine, 350, p. 2129-213 9 (2004)

Non-patent literature 3: British Journal of Cancer, 93, p. 355-363 (2005) Non-patent literature 4: Clinical Cancer Research, 11 (12), p.4289-4294 (2005) Non-patent literature 5: Nature Reviews, 3, p. 749-761 (2004)

Non-Patent Document 6: BioTechniques, 20, p. 240-248 (1996)

Non-patent literature 7 Analytical Biochemistry, 333 (2), p. 246-255 (2004) Non-patent literature 8: Nucleic Acids Research, 28, p. 2752- 3761 (2000) Non-patent literature 9: Clinical Chemistry, 46, p . 24-30 (2000)

Disclosure of the invention

Problems to be solved by the invention

[0005] As described above, S is being studied for the analysis of deletion mutations with a constant deletion pattern, and in the detection of atypical deletion mutations with a non-constant deletion pattern, depending on the individual case, after sequence analysis and nucleic acid amplification There is a problem that it is difficult to perform easily and quickly because it requires a complicated and complicated process such as high-performance electrophoretic analysis (for example, capillary single electrophoresis) and mass spectrometry using TOF-MS. Furthermore, if the specimen is cancer tissue, the gene derived from cancer cells that are mixed in the presence of the majority of normal cells, rather than the mixed rate of 100% or 50%, as in normal gene polymorphism analysis Need to be detected. For example, it was difficult to detect mutant genes present at a mixing rate of 5%.

[0006] An object of the present invention is to provide epidermal growth factor receptor (EGFR) residues with various mutation patterns and short deletion sequences, so-called loss patterns are not constant. To provide a simple, rapid, and sensitive screening method useful for predicting the effects of anticancer drugs (EGFR inhibitors), epidemiological studies, and disease predictions for somatic cell deletion mutations in exon 19 is there. Means for solving the problem

[0007] The present inventors diligently studied and developed a novel technique capable of detecting even a defective mutant gene mixed at a rate of 5% in a normal EGFR gene. Furthermore, a new technology has been developed to quickly perform the detection described above in a closed and closed system in the same reaction system. In other words, it is closed with a single tube, preventing the risk of cross-contamination in a homogeneous system, allowing multiple samples to be measured simultaneously in a short period of 90 minutes, and detecting only the presence of 5% of the defective mutant gene in the normal gene A possible high-sensitivity detection method and reagent kit were developed to complete the present invention.

[0008] That is, the first invention of the present invention relates to a method for detecting the presence or absence of a deletion mutation of exon 19 of EGFR gene contained in a sample, characterized in that it comprises the following steps: Deletion of EGFR gene Amplifying the target region in the sample using at least one deletion sequence primer that can be annealed to the junction region and a primer opposite to the deletion sequence primer, where the nucleotide sequence at the 3 ′ end of the deletion sequence primer Is a base sequence consisting of 2 to 5 bases derived from the 3 'non-deletion site adjacent to the defective junction, and the primer facing the defective sequence primer can be annealed to both the defective mutation and the normal gene. Detecting a deletion mutation based on the presence or absence of an amplification product.

[0009] In addition, in the first invention of the present invention, the deletion mutation is further performed in the same reaction system as the amplification reaction by using at least two probes including a probe for recognizing a defective portion and a probe for recognizing a non-defective portion. It may be detected.

[0010] In addition, in the first invention of the present invention, the deletion sequence ply selected from the primers having any one of the base sequences set forth in SEQ ID NOs: 6 to 10 in the sequence listing is not limited. It is preferable to use a primer that opposes the deletion sequence primer having the base sequence described in SEQ ID NO: 11 in the sequence table and / or a probe having the base sequence described in SEQ ID NO: 16 to 17 in the sequence list. [0011] A second invention of the present invention relates to a composition for the method of the first invention of the present invention, characterized by comprising:

(1) Deletion sequence primer that can be annealed to the defective junction region of the EGFR gene, where the 3 'terminal nucleotide sequence of the deletion sequence primer is derived from the non-deletion site on the 3' side adjacent to the deletion junction Is a base sequence consisting of 2 to 5 bases,

(2) A primer opposite to the primer for the defective sequence of (1), wherein the primer is a primer capable of annealing to both a defective mutation and a normal gene, and

(3) DNA polymerase.

[0012] In the second invention of the present invention, (4) a probe that recognizes a defective site, (5) a probe that recognizes a non-defective site, and (6) a probe that is cleaved when hybridized to a target sequence It may contain enzymes,

[0013] In addition, in the second invention of the present invention, the deletion sequence ply selected from primers having the base sequences set forth in any one of SEQ ID NOs: 6 to 10 in the sequence listing is not particularly limited. And a primer having a nucleotide sequence described in SEQ ID NO: 11 in the sequence listing and a primer opposite to the primer for the defective sequence and / or a probe having the base sequence described in SEQ ID NO: 16 to 17 in the sequence listing. Masle.

[0014] A third invention of the present invention relates to a kit for the method of the first invention of the present invention, characterized by comprising:

(1) A deletion sequence primer that can be annealed to the defective junction region of the EGFR gene, where the 3 ′ terminal nucleotide sequence of the deletion sequence primer is derived from the non-deletion site on the 3 ′ side adjacent to the deletion junction A base sequence consisting of 2 to 5 bases, and

(2) A primer that is opposite to the primer for the defective sequence of (1), wherein the primer is a primer that can be annealed to both a defective mutation and a normal gene.

In the third invention of the present invention, it may further contain (3) a probe for recognizing a deficient site and (4) a probe for recognizing a non-deficient site.

[0015] In addition, in the third invention of the present invention, the deletion sequence ply selected from primers having a base sequence set forth in any one of SEQ ID NOs: 6 to 10 in the sequence listing in the third invention of the present invention. And a primer that opposes the deletion sequence primer having the nucleotide sequence set forth in SEQ ID NO: 11 in the sequence listing and / or a probe having the nucleotide sequence set forth in SEQ ID NO: 16 to 17 in the sequence listing. Masle.

The invention's effect

[0016] According to the present invention, the somatic cell deletion mutation in exon 19 of the EGFR gene is useful in terms of anticancer drug (EGFR inhibitor) effect prediction, epidemiological investigation, and disease prediction, which is convenient, quick and highly sensitive. Screening is possible.

Brief Description of Drawings

[0017] FIG. 1-1 shows a pattern of deletion mutations in exon 19 of the BGFR gene.

FIG. 1-2 shows a pattern of deletion mutations in exon 19 of the EGFR gene.

FIG. 2 is a graph showing calculation and plotting of HEX fluorescence intensity and H EX / FAM value per FAM fluorescence intensity in each sample of Example 5.

BEST MODE FOR CARRYING OUT THE INVENTION

[0018] As used herein, a deletion mutation refers to a mutation in which a part of DNA is missing from DNA having a normal sequence.

[0019] In this specification, a defective site refers to a DNA site that is lacking in the above-described deletion mutation.

[0020] In the present specification, the non-deletion site means a site other than the above-mentioned defect site.

In the present specification, a defective junction refers to a portion between bases adjacent to each other on both sides of a defective site in a gene having a defective mutation. That is, it refers to the junction between non-deficient sites in a defective mutation.

[0022] In the present specification, a defective junction region refers to a gene having a defective mutation.

, Refers to a region containing bases adjacent to both sides of the defect site. That is, it refers to a region containing bases adjacent to both sides of the junction between non-deficient sites (ie, a defective junction) in a defective mutation.

[0023] In this specification, the primer opposite to the deletion sequence primer refers to a primer that constitutes a primer pair used in the nucleic acid amplification reaction together with the deletion sequence primer. Hereinafter, the present invention will be described in detail.

(A) EGFR gene deletion mutation detection method of the present invention

The detection method of the present invention comprises:

A method for detecting the presence or absence of a deletion mutation in exon 19 of an EGFR gene contained in a sample, wherein at least one primer for a deletion sequence that can be annealed to a deletion junction region of the EGFR gene, wherein 'The base sequence at the end is a base sequence IJ consisting of 2 to 5 bases derived from the non-deletion site adjacent to the deletion junction, and a primer opposite to the deletion sequence primer, where the primer is a deletion mutation It is a primer that can be annealed to both normal genes and normal genes, and is characterized by amplifying a target region in a sample. Examples of the amplification product detection method include agarose gel electrophoresis and a detection method using a probe.

It is also possible to detect a defective mutation in the same reaction system as the amplification reaction by using at least two probes for identifying a defective site and a probe for identifying a non-defective site.

Examples of the deletion mutation of exon 19 of the EGFR gene to be detected in the present invention include a deletion mutation appearing at codons 746 to 752 of exon 19 of the EGFR gene. Preferred examples of such detection targets include the deletion mutations described in the previous non-patent documents:! To 4, more preferably the deletion mutations described in the non-patent documents 1 and 2 (FIG. 1). Is illustrated. In Figure 1, deletions Del_la, Del-lb, Del_3, Del_4, Del_5, and Del-2 are deletions of bases 2235 to 2249 (deletions of amino acids E746 to A750) and deletions of bases 2236 to 2250, respectively. (Deletion of amino acids E746 to A750), deletion of bases 2239 to 2247 (deletion of amino acids L747 to E749, substitution of A750P), deletion of bases 2240 to 2257 (deletion of amino acids L74 7 to S752, P753S Substitution), deletion of bases 2238 to 2255 (deletion of amino acids L747 to S752, substitution of E746V), deletion of bases 2254 to 2277 (deletion of amino acids S752 to I759). Figure 1 and the above base numbers are the numbers when the start codon ATG A of the human EGFR gene is 1, and the amino acid numbers are the numbers when the methionine (Met) encoded by the start codon is 1. is there. In addition, one embodiment of the method of the present invention is characterized in that at least one pair of primers for detecting the above-mentioned deletion mutation, at least two probes that recognize a deletion site, and a non-deletion site are used. Another aspect of the method of the present invention is characterized in that a nucleic acid amplification reaction and signal detection are simultaneously performed in the same reaction system, and the presence or absence of a deletion mutation is detected rapidly and with high sensitivity.

[0025] As the primer used in the method of the present invention, any primer that does not substantially amplify the normal EGFR gene and can substantially amplify at least one kind of deletion mutation can be preferably used. Although there is no particular limitation, for example, at least one of the primers used in the method of the present invention can be annealed to the defective junction region of the EGFR gene, and the nucleotide sequence at the 3 ′ end of the primer is adjacent to the defective junction. It is selected from the nucleotide sequences derived from the 3 ′ non-deficient site. The base sequence at the 3 ′ end of the primer is preferably 2 to 5 bases derived from the non-deleted site on the 3 ′ side adjacent to the defective junction, particularly preferably 2 to 3 bases. In addition, two or more kinds of the primers can be used for nucleic acid amplification reaction in the same reaction system. This is preferable in that two or more types of deletion mutations can be detected in the same reaction system. When using two or more kinds of the above primers, the positions where the 5 'ends of the primers are annealed match in order to identify the chain length of the missing base sequence by determining the chain length by agarose gel electrophoresis or the like. Even if you set the primer like so, As said primer, what has the base sequence of any one of sequence number 6-: 10 of a sequence table can be used conveniently, for example.

[0026] The other primer used in the method of the present invention is not particularly limited as long as it can be annealed to both a deletion mutation of the gene and a normal gene. Primers designed to amplify DNA from, for example, paraffin sections are difficult. As the primer, a primer that is set so that the chain length of the amplification product obtained by the nucleic acid amplification reaction is less than or equal to lOObp is preferred, and the chain length of the amplification product is preferably set to be less than 87bp. It is a primer that has been used. As the primer, for example, a primer having a base sequence described in SEQ ID NO: 11 in the sequence listing can be preferably used. In the method of the present invention, the target defective gene can be detected with high sensitivity by the above primer pair.

[0027] In addition to the above matters, when setting the primer used in the method of the present invention, a known document such as Uchiumi et al. (Protein, Nucleic Acid, Enzyme, No. 35, p3157 to p3163, 1990) You can refer to the guidelines for preparing primers described in the year).

[0028] In the present invention, the DNA fragment amplified by the method of the present invention may be detected using a probe.

[0029] The probe used in the method of the present invention is not particularly limited as long as it has a base sequence that recognizes a defective site and a base sequence that recognizes a non-defective site, but a sequence that is complementary to the target sequence. Those having are preferred. The probe is preferably, for example, a probe that is cleaved by a specific nucleolytic enzyme when hybridized to a target sequence. Such probes include TaqMan ™ probes and DNA-RNA-DNA chimeric probes. A particularly preferred probe is a DNA-RNA-DNA chimera probe. The chain length of the RNA portion of the chimeric probe is not particularly limited, but it is preferably 1 to 5 bases from the viewpoint of the stability of the probe, more preferably 1 base. The chain length of the entire probe is preferably 6 to 30 bases, more preferably 10 to 15 bases.

Of the above probes, as a probe for recognizing a defective site, it is preferable to select a region that is commonly deleted in various deletion mutations. A part of the sequence of the probe may contain a non-deficient site, but it is preferable to avoid annealing of the amplified deficient DNA and the non-deficient site of the probe within the range of amplification temperature conditions. The non-deficient site of the probe is preferably 0 to: 12 bases, more preferably 0 to 6 bases. Although there is no particular limitation, for example, the base sequence described in SEQ ID NO: 16 in the sequence listing is preferred.

[0030] Further, as the probe for recognizing a non-deletion site, a probe in which a region that does not receive a defect in common among various defects can be suitably used. Although not particularly limited, for example, those having the base sequence set forth in SEQ ID NO: 17 in the sequence listing are preferred. [0031] In addition, when there is no region common to other deletion mutations in the deletion site, or there is a deletion mutation in which the chain length of the common deletion region is not sufficient for setting a probe, For deletion mutations, the above-mentioned probe for recognizing a deficient site can be used as a probe for recognizing a non-deficient site, and a probe for recognizing a non-deficient site can be used as a probe for recognizing a deficient site.

Although there is no particular limitation, for example, the deletion mutation of exon 19 of the EGFR gene shown in Fig. 1 is explained as an example. Deletion sites of deletion sequences Del_la, Dellb, Del-3, Del_4, Del-5 The probe DF (SEQ ID NO: 16) that recognizes can be used as a probe for recognizing a non-deficient site with respect to the defective ligand IjDel-2. In addition, the probe NH that recognizes the non-deletion site of the deleted sequences Delia Dellb, Del—3, Del—4, Del—5 (Kami no. It can be used as a recognition probe.

[0032] Furthermore, in the method of the present invention, although not particularly limited, gene amplification methods often used in the art such as PCR method, ICAN method, LAMP method, SDA method and the like can be used. A particularly preferred method is, for example, the PCR method.

[0033] Further, examples of the real-time detection method that can be used in the present invention include TaqMan method, Scoi ^ ion method, Cycling probe method, noise hybridization probe method, and Cycleave method. Although there is no particular limitation, the Cycleave method is preferred. In the Cycleave method, a hybrid is formed between the amplification product and the RNA-containing probe, and the RNA sequence of the probe is cleaved by RNaseH in the reaction solution only when the base sequence of the amplification product and the probe is a perfect match. The part is not cut. Taking advantage of this, when a probe labeled with two or more fluorescent dyes having different wavelengths and causing interference of fluorescence between the fluorescent dyes is used as the probe, the probe is cleaved. Therefore, since the fluorescence intensity of the fluorescent substance changes, the presence or absence of mutation can be determined. For example, 6_FAM (6_carboxy fluorescein) and HEX (Hexachloro_6_carboxyfluoresceiru), 6—FAM and ROX (6-carboxy-X-rhodamine, Combinations such as ABI) can be used.

[0034] Further, the probe has a function of quenching a fluorescent substance and fluorescence emitted from the fluorescent substance. The substance may be labeled with an appropriate interval, for example, Eclipse (Epoch Biosciences) or DABCYL (4_dimethylaminoazobenzene-4′-sulfone). In such a probe, a part of the fluorescence emitted by the fluorescent substance is converted into thermal energy by the quenching substance in an interactive state. However, when the distance between the fluorescent substance and the quenching substance is increased due to the cleavage, the above action is achieved. Is eliminated, and the detected fluorescence signal intensity increases.

[0035] In the Cycleave method, two or more types of probes can be used in the same reaction system. In such a case, each probe is labeled with a fluorescent dye having a different wavelength, and the presence or absence of mutation can be determined more clearly from the ratio of the change in fluorescence intensity. For example, when a probe that detects a defective site in a target nucleic acid and a probe that detects a normal site are labeled with fluorescent substances having different wavelengths and are used in the Cycleave method, two probes are present if a defect exists. Among them, an increase in the fluorescence signal intensity due to the probe corresponding to the defective site is not detected, and only an increase in the fluorescence signal intensity due to the other probe corresponding to the normal site can be detected.

[0036] (B) Composition for the method of the present invention

The composition of the present invention comprises:

A composition for the method described in (A) above,

(1) Deletion sequence primer that can be annealed to the defective junction region of the EGFR gene, where the 3 'terminal nucleotide sequence of the deletion sequence primer is derived from the non-deletion site on the 3' side adjacent to the deletion junction Is a base sequence consisting of 2 to 5 bases of

(3) It contains DNA polymerase.

Furthermore,

(4) a probe for identifying a defect site, and

(5) It is characterized by containing a probe that recognizes a non-deficient site.

[0037] In the composition of the present invention, although not particularly limited, for example, a deletion sequence ply selected from primers having a base sequence described in any one of SEQ ID NOs: 6 to 10 in the Sequence Listing. And a primer opposite to the deletion sequence primer having the nucleotide sequence set forth in SEQ ID NO: 11 in the Sequence Listing.

Furthermore, it may contain a probe having the base sequence described in any one of SEQ ID NOS: 16 and 17 in the sequence listing.

The probe may be labeled with the label described in (A) above.

[0038] The DNA polymerase contained in the composition of the present invention is not particularly limited. For example, raq (Thermus aquaticus) which is generally used for DNA amplification is preferred. DNA polymerase, Pfu (Pyrococcus furiosus-derived DNA polymerase, Tli (Thurmococcus litoralis) -derived DNA polymerase, KOD (Thermococcus kodakaraensis) -derived DNA polymerase, Bca (Bacillus caldotenax-derived DNA polyhusose, Bst (Geobacillus stearothermophilus; -derived DNA polymerase) And DNA polymerase in which two or more of the above DNA polymerases are mixed, and Taq-derived DNA polymerase is more preferable.

[0039] In addition, the composition of the present invention may contain an enzyme for cleaving the probe when the composition can be used in the Cycling probe method or the Cycleave method. Examples include, but are not limited to, RNaseH (ribonuclease H). RNaseH is more preferably derived from archaea, and in particular, RNaseH (Tli RNaseH), which can be prepared by the method described in WO 02/22831, is preferably used. Further, the composition may contain a nucleic acid amplification reaction reagent and the like.

[0040] (C) Kit for the method of the present invention

The kit of the present invention comprises

A kit for the method described in (A) above,

(1) Deletion sequence primer that can be annealed to the defective junction region of the EGFR gene, where the 3 'terminal nucleotide sequence of the deletion sequence primer is derived from the non-deletion site on the 3' side adjacent to the deletion junction And a base sequence consisting of 2 to 5 bases, and

(2) Primer opposite to the deletion sequence primer in (1), where the primer is missing It is a primer that can be annealed to both a loss mutation and a normal gene.

Furthermore,

(3) a probe for identifying a defect site, and

(4) A probe that recognizes a non-deficient site is contained.

[0041] In the kit of the present invention, although not particularly limited, for example, a primer for a deletion sequence selected from a primer having a base sequence described in SEQ ID NO: 6 to 10 in the sequence listing and SEQ ID NO: in the sequence listing A primer that opposes the deletion sequence primer having the base sequence according to 11, may be used.

Furthermore, it may contain a probe having the base sequence described in any one of SEQ ID NOS: 16 and 17 in the sequence listing. Further, the probe may be labeled with the label described in (A) above.

[0042] As the DNA polymerase contained in the kit of the present invention, those exemplified in the above (2) can be preferably used.

[0043] The kit of the present invention may contain the enzyme exemplified in the above (2). Furthermore, it may contain nucleic acid amplification reaction reagents.

[0044] By using the composition of the present invention and / or the kit of the present invention, the presence or absence of a deletion mutation in the EGFR gene can be easily detected.

[0045] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the scope of the examples.

Example 1

[0046] First, control template DNA and a primer for detecting a defective sequence were synthesized. That is, a normal sequence control template DNA (SEQ ID NO: 1 in the sequence listing) was synthesized from a nucleic acid sequence from the 1648th 56th to the 164948th of a human EGFR gene array IJ (GenBank Accession No. AF288738) by a DNA synthesizer. This cattle IJ contains a part of human EGFR gene exon 19 (SEQ ID NO: 18 in the sequence listing). In addition, EGFR exon 19 deficiency reported in Paez et al., Science, 304, 1497-1500 (2004). IjDel-la, Del-lb, Del-3, Del-4 amino acid sequence information And the above human EGFR gene sequence From the information, CTDel—la (SEQ ID NO: 2 in the sequence listing), CTDel—lb (SEQ ID NO: 3 in the sequence listing), CTDel—3 (SEQ ID NO: 4 in the sequence listing), and CTDel—4 (SEQ ID NO: 5 in the sequence listing). ) Were synthesized with a DNA synthesizer and used as a defective sequence control template DNA. The obtained normal sequence control template DNA and defective sequence control template DNA were prepared to be lOOfgZ 1 with sterilized distilled water, respectively.

[0047] Next, as an upstream primer for detection of deletion sequences Del_la, Del-lb, Del_3, Del_4 of EGFR exon 19, FlDla (SEQ ID NO: 6 in the sequence listing), F2Dlb (SEQ ID NO: 7 in the sequence listing), F3D3 (SEQ ID NO: 8 in the Sequence Listing), F4D4 (SEQ ID NO: 9 in the Sequence Listing) and F5D5 (SEQ ID NO: 10 in the Sequence Listing) were respectively synthesized by a DNA synthesizer. In addition, RV (SEQ ID NO: 11 in the sequence listing) was synthesized by a DNA synthesizer as a downstream primer for detection common to the deletion sequence and normal sequence of EGFR exon 19. Furthermore, as an upstream primer for detection of deletion sequences Del-la, Del-lb, Del-3 and Del-4 of EGFR exon 19, only the 3 'terminal monobasic group differs from the normal sequence F1-2Dla (sequence in the sequence listing) No. 12), F2—2Dlb (SEQ ID NO: 13 in the sequence listing), F3—2D3 (SEQ ID NO: 14 in the sequence listing), and F4—2D4 (SEQ ID NO: 15 in the sequence listing) were also synthesized by a DNA synthesizer. . Figure 1 shows the position of the downstream primer RV. In Figure 1, the position of each upstream primer is underlined.

Example 2

[0048] High-sensitivity detection of the EGFR exon 19-deficient gene by multiplex PCR using the defective sequence primer prepared in Example 1 was studied. First, a DNA having a cage shape was prepared as follows. That is, defect Hai歹IjCTDel- la is to be 5 mole ^_0/0 for all control template, the lOOfg / μ Ι CTDel- 1 a deficiency sequence control template DNA solution prepared in Example 1 lOOfg / μ 1 Normal sequence control template Dilute with template DNA solution to make 5 mol% 〇061-la-deficient sequence control template DNA solution. Similarly, CTDel-lb solution, CTDel-3 solution, and CTDel 4 solution were also diluted with lOOfg / μΙ normal sequence control template DNA solution, respectively, to obtain 5 mol% deletion sequence control template DNA solution.

[0049] Each 5 mol% deletion sequence control template prepared above. Deletion in DNA solution Sequence DNA was detected by multiplex PCR. The composition of the reaction mixture, 10 XP CR buffer (manufactured by Takara Bio Inc.), the normal sequence control template DNA co cement roll template DNA or l OOfg containing 5 deficiency sequences mole ^_0/0 lOOfg as铸型, and 1. 25U Using TaKaRa Taq DNA Polymerase (Takara Bio Inc.), final concentration force ^s 0.2 mM dNTP, 2 mM MgCl, 0.2 μ μ FlDla primer, 0.2

2

It prepared so that it might become (micro | micron | mu) F2Dlb primer, 0.2 micromicron F3D3 primer, 0.2 micromol F4D4 primer, 0.2 micromicron F5D5 primer, 0.2 micromicron RV primer. The reaction volume is 25 μ1. The PCR amplification system uses a thermal cycler MP (Takaranoku), and the reaction conditions are 95. C 30 deficiency, 60. C 30 deficiency, 72. The reaction was a 35-cycle reaction with a C cycle of 1 cycle. After completion of the reaction, the amplified product was electrophoresed with 3% NuSieve3: lagarose (Takara Bio Inc.) and stained with ethidium bromide to detect the amplified product.

[0050] As a result, only the amplification bands corresponding to the amplified chain lengths specific to each of the defective sequence control DNAs were detected in all the samples in which the four types of 5 mol% defective sequence control templates were in the cocoon shape. It was. In addition, in the sample in which the normal sequence control template DNA was in a saddle type, an 87 bp amplified band corresponding to the normal amplified chain length could not be confirmed. Based on this fact, it was confirmed that the defective sequence in the sample can be accurately amplified by the method of the present invention.

Example 3

[0051] EGFR exon 19 deletion gene detection by multiplex PCR method using a primer for a deletion sequence that differs from the normal sequence only at the 3 'end reported as ARMS method and the method of the present invention. Comparison · Reviewed.

That is, the upstream primer for EGFR exon 19 deletion sequence, which differs from the normal sequence of EGFR exon 19 only in the 1 ′ base at the 3 ′ end prepared in Example 1, Fl-2Dla, F2-2Dlb, F3-2 D3, F4-2D4, and F5-2D5 was used to detect defective sequence DNA in a 5 mol% defective sequence control template DNA solution. PCR conditions and subsequent detection of amplification products were performed under the same conditions as in Example 2 except for the upstream primer for detecting the defective sequence to be used. [0052] As a result, when the 5 mol% deletion sequence control template DNA of Del-1 and Del-lb was used as a saddle type, only an amplified product of 88 bp corresponding to the amplified chain length of normal sequence DNA as a saddle type was obtained. Was detected. In Del-4, there is an amplification product of 88 bp corresponding to the amplified strand length of normal sequence DNA and a 70 bp amplified product corresponding to the amplified strand length specific to Del-4 deficient sequence DNA. Detected at a rate of.

Based on the above results and the results of Example 2, the highly defective detection of 1D JDNA in the presence of a large number of normal sequence DNAs. It was confirmed that a primer having a terminal sequence different from the normal sequence by 2 to 3 bases was effective.

Example 4

[0053] The detection limit of detection of EGFR exon 19-deficient gene in the detection method of the present invention was examined.

That is, the 5 mol% deletion sequence control template DNA solution of lOOfgZ 1 prepared in Example 2 was further diluted with a normal sequence control template DNA solution of lOOfg / μ 1 to obtain 1, 0.1, 0.01 mol. % Deficient sequence control template DNA solution was prepared. Using each of these DNAs, lOOfg was subjected to PCR amplification under the same conditions as in Example 2, and the detection sensitivity for defective DNA in the presence of normal sequence DNA was examined. As a result, it was possible to detect the defectively distributed control template DNA up to 1 mol% for Del_la and 061_1, and up to 0.1 mol% for Del-3 and Del-4.

Example 5

[0054] Simple detection using the cycling probe method of EGFR exon 19-deficient gene amplification product by deletion-specific primer multiplex PCR was examined. As a probe for detection of EGFR exon 19 deletion sequence used for detection, FAM (manufactured by Applied Biosystems) is labeled at the 5 'end, and Eclipse Quenchia (manufactured by Epoch) is labeled at the 3' end. SEQ ID NO: 16) and probe NH (SEQ ID NO: 17 in the sequence listing) each labeled with HEX (Applied Systems) and labeled with Eclipse Quencher at the 3 ′ end were synthesized by a DNA synthesizer.

[0055] Using the above probe, E by deletion-specific primer multiplex PCR Simple detection of the amplified product of GFR exon 19-deficient gene using the cycling probe method was performed. The composition of the reaction solution is 10 X PCR buffer (Takara Bio Inc.), 100 fg of control template DNA containing 5 mol% deletion sequence as a cage, l OOfg normal sequence control template DNA or l OOng normal genomic DNA, 1. Using 25U TaKa Ra Taq DNA Polymerase (Takara Bio Inc.) and 50U Tli RNaseH II (Takara Bio International Publication No. 02Z22831 pamphlet), final concentrations of 0.2 mM dNTP, 2 mM MgCl 2, 0.2 μΜ Fl D la primer, 0.2 μM F2D lb ply

2

, 0.2 μΜ F3D3 primer, 0.2 μΜ F4D4 primer, 0.2 μM F5D5 primer, 0.2 μΜ RV primer, 0.2 μ μ probe ΝΗ, 0.2 μΜ probe Prepared to be DF. The reaction volume is 25 μ1. The 7500 system (Applied Biosystems) was used for PCR amplification and fluorescence intensity detection. PCR was performed in a 40-cycle reaction where 15 cycles of 95 ° C, 40 cycles of 60 ° C, and 30 cycles of 72 ° C were used. Simultaneously with PCR, the fluorescence intensity of FAM and HEX was measured by ABI7500 system.

[0056] As a result, the increase in the fluorescence intensity of HEX caused by cleavage of the probe NH in the non-defect region by RNaseH was remarkably observed in all reactions using 5 mol% deletion sequence control template DNA. It was. On the other hand, the increase in the fluorescence intensity of FAM caused by the cleavage of probe DF by RNaseH in the region subject to the defect in common was hardly confirmed in all reactions in which the 5 mol% -deficient control template DNA was trapezoidal. On the other hand, in the reaction using normal sequence control template DNA and normal genomic DNA as a saddle type, the increase in fluorescence intensity caused by the cleavage of probe DF by RNaseH and the cleavage of probe NH by RNaseH was almost confirmed. Based on this fact, it was confirmed that real-time detection was possible using the probe.

[0057] Furthermore, in order to clarify the difference in fluorescence intensity between the normal sequence DNA and the mutant sequence DNA, the HEX fluorescence per FAM fluorescence intensity in each sample was clarified. Intensity and HEXZFAM values were calculated and plotted. Figure 2 shows the results. Here, N is the HEX / FAM value when the normal sequence control template DNA is a cocoon type, and N1 to N5 are the HEX / FAM values when the normal genomic DNA is a cocoon type, Dell-la, Dell-lb, Del—3, and Del_4 indicate the HEXZFAM values when the deletion sequence control template DN A is a vertical type. As shown in Fig. 2, the normal sequence control template DNA and 5 types of normal genomic DNA showed a very low HEX / FAM value of 0.5 or less in all cases. The HEXZFAM value is 5.8 to 52.5 for the mol% deletion sequence control template DNA, and the sample containing the deletion sequence control template DNA is 10 times higher than the sample containing only the normal sequence DNA. Indicated. Thus, it was shown that the presence of the EGFR exon 19 deletion sequence can be clearly determined by using the HEXZFAM value as an index.

As described above, by combining the cycling probe method and the defective sequence-specific primer multiplex PCR method, even in the presence of normal genes, 5 mol% of EGFR exon 19-deficient genes can be detected, which is highly sensitive. Thus, it was confirmed that the simple detection method using Singnotube is effective.

Industrial applicability

[0058] According to the present invention, there is provided a simple, rapid and highly sensitive detection method for a somatic deletion mutation in exon 19 of the EGFR gene, in which the deletion pattern is not constant. The detection method is useful in predicting the effects of anticancer agents, epidemiological studies, and disease prediction.

Sequence listing free text

[0059] SEQ ID NO: l: Control template DNA

SEQ ID NO: 2: Control template DNA CTDel-la

SEQ ID NO: 3: Control template DNA CTDel-lb

SEQ ID NO: 4: Control template DNA CTDel-3

SEQ ID NO: 5: Control template DNA CTDel-4

SEQ ID NO: 6: PCR primer FlDla to amplify a deletion mutant "Del- la

SEQ ID NO: 7: PCR primer F2Dlb to amplify a deletion mutant "Del-lb"

SEQ ID NO: 8: PCR primer F3D3 to amplify a deletion mutant "De 3"

SEQ ID NO: 9: PCR primer F4D4 to amplify a deletion mutant "Del-4"

SEQ ID NO: 10: PCR primer F5D5 to amplify a deletion mutant "De 5" SEQ ID NO: 11: PCR primer RV to amplify a deletion mutant of EGFR exon 19 SEQ ID NO: 12: PCR primer Fl-2Dla to amplify a deletion mutant

SEQ ID N〇13: PCR primer F2-2Dlb to amplify a deletion mutant "Del-lb"

SEQ ID N〇14: PCR primer F3-2D3 to amplify a deletion mutant "Del-3"

SEQ ID N〇15: PCR primer F4-2D4 to amplify a deletion mutant "De 4"

SEQ ID NO: 16: Chimeric oligonucleotide probe DF to detect the DNA fragment of human mutant type EGFR exon 19.nucleotides 4 is a ribonucleotides- other nucleoti des are deoxynbonucleotides

SEQ ID N〇17: Chimeric oligonucleotide probe NH to detect the DNA fragment of human mutant type EGFR exon 19. nucleotides 5 is a ribonucleotides-other nucleoti des are deoxynbonucleotides

Claims

The scope of the claims

[1] A method for detecting the presence or absence of an EGFR gene exon 19 deletion mutation in a sample, comprising the following steps:

Amplifying the target region in the sample using at least one defective sequence primer that can be annealed to the defective junction region of the EGFR gene and a primer opposite to the defective sequence primer, where 3 ′ of the defective sequence primer The terminal base sequence is a base sequence consisting of 2 to 5 bases derived from the 3 ′ non-deletion site adjacent to the deletion junction, and the primer opposite the deletion sequence primer is either a deletion mutation or a normal gene. Are also primers that can be annealed; and detecting defective mutations based on the presence or absence of amplification products.

[2] The deletion mutation is further detected in the same reaction system as the amplification reaction using at least two probes including a probe that recognizes a defective site and a probe that recognizes a non-defective site. The method described.

[3] A deletion sequence primer selected from a primer having any one of the base sequences described in SEQ ID NO: 6 to 10 in the sequence listing and the above-mentioned deletion sequence primer having the base sequence described in SEQ ID NO: 11 in the sequence listing; The method according to claim 1, wherein an opposing primer is used.

[4] The method according to claim 2, wherein a probe having the base sequence described in any one of SEQ ID NOS: 16 and 17 in the sequence listing is used.

[5] The composition for the method according to claim 1, comprising:

(1) A primer for a defective sequence that can be annealed to a defective junction region of the EGFR gene, where the base sequence at the 3 'end of the primer for the defective sequence is derived from the non-defective site on the 3' side adjacent to the defective junction Is a base sequence consisting of 2 to 5 bases,

(3) DNA polymerase.

[6] In addition,

(4) a probe that recognizes the defect site, (5) a probe that recognizes a non-deficient site, and

6. The composition according to claim 5, further comprising an enzyme for cleaving the probe when hybridized to a target sequence.

[7] A deletion sequence primer selected from a primer having a base sequence described in any one of SEQ ID NOs: 6 to 10 in the sequence listing and a primer for the deletion sequence having a base sequence described in SEQ ID NO: 11 in the sequence listing; Claims containing opposing primers

5. The composition according to 5.

[8] The composition according to [6], comprising a probe having the base sequence described in SEQ ID NO: 16 or 17 in the sequence listing.

[9] The kit for the method according to claim 1, comprising:

(1) A primer for a defective sequence that can be annealed to a defective junction region of the EGFR gene, where the base sequence at the 3 'end of the primer for the defective sequence is derived from the non-defective site on the 3' side adjacent to the defective junction A base sequence consisting of 2 to 5 bases, and

[10] In addition,

(3) a probe for recognizing a defect site, and

(4) The kit according to claim 9, comprising a probe that recognizes a non-deficient site.

[11] SEQ ID NO: 6 to 10 in the sequence listing: a deletion sequence primer selected from primers having the base sequence described in any of 10 and the deletion sequence primer having the base sequence described in SEQ ID NO: 11 in the sequence listing; 10. The kit according to claim 9, wherein the kit contains opposing primers.

[12] The kit according to claim 10, comprising a probe having the base sequence of any one of SEQ ID NOS: 16 and 17 in the sequence listing.