WO2006070667A1 - Procede de detection d'une mutation dans le gene egfr et kit de detection - Google Patents

Procede de detection d'une mutation dans le gene egfr et kit de detection Download PDF

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WO2006070667A1
WO2006070667A1 PCT/JP2005/023486 JP2005023486W WO2006070667A1 WO 2006070667 A1 WO2006070667 A1 WO 2006070667A1 JP 2005023486 W JP2005023486 W JP 2005023486W WO 2006070667 A1 WO2006070667 A1 WO 2006070667A1
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sequence
primer
defective
deletion
probe
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PCT/JP2005/023486
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English (en)
Japanese (ja)
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Masamitsu Shimada
Fumitsugu Hino
Ikunoshin Kato
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Takara Bio Inc.
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Priority to JP2006550710A priority Critical patent/JPWO2006070667A1/ja
Publication of WO2006070667A1 publication Critical patent/WO2006070667A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • 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.
  • Non-Patent Document 5 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).
  • deletion mutation analysis has been carried out by sequencing and high performance electrophoresis.
  • Patent Document 9 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)
  • 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.
  • EGFR epidermal growth factor receptor
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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,
  • 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,
  • 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.
  • 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:
  • 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
  • the third invention of the present invention may further contain (3) a probe for recognizing a deficient site and (4) a probe for recognizing a non-deficient site.
  • 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 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.
  • 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.
  • a deletion mutation refers to a mutation in which a part of DNA is missing from DNA having a normal sequence.
  • a defective site refers to a DNA site that is lacking in the above-described deletion mutation.
  • the non-deletion site means a site other than the above-mentioned defect site.
  • 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.
  • a defective junction region refers to a gene having a defective mutation.
  • a region containing bases adjacent to both sides of the defect site 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.
  • 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.
  • the present invention will be described in detail.
  • 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.
  • the amplification product detection method include agarose gel electrophoresis and a detection method using a probe.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a primer having a base sequence described in SEQ ID NO: 11 in the sequence listing can be preferably used.
  • the target defective gene can be detected with high sensitivity by the above primer pair.
  • the DNA fragment amplified by the method of the present invention may be detected using a probe.
  • 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 TM 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.
  • the 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • Eclipse Epoch Biosciences
  • DABCYL 4_dimethylaminoazobenzene-4′-sulfone
  • 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.
  • 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.
  • composition of the present invention comprises:
  • 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
  • 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.
  • 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.
  • the DNA polymerase contained in the composition of the present invention is not particularly limited.
  • raq Thermus aquaticus
  • 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.
  • 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.
  • an enzyme for cleaving the probe 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.
  • the composition may contain a nucleic acid amplification reaction reagent and the like.
  • the kit of the present invention comprises
  • 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
  • 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.
  • DNA polymerase contained in the kit of the present invention those exemplified in the above (2) can be preferably used.
  • the kit of the present invention may contain the enzyme exemplified in the above (2). Furthermore, it may contain nucleic acid amplification reaction reagents.
  • 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.
  • 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).
  • 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
  • CTDel—4 SEQ ID NO: 5 in the sequence listing.
  • a DNA having a cage shape was prepared as follows. That is, defect Hai ⁇ IjCTDel- la is to be 5 mole 0/0 for all control template, the lOOfg / ⁇ ⁇ CTDel- 1 a deficiency sequence control template DNA solution prepared in Example 1 lOOfg / ⁇ 1 Normal sequence control template Dilute with template DNA solution to make 5 mol% ⁇ 061-la-deficient sequence control template DNA solution. Similarly, CTDel-lb solution, CTDel-3 solution, and CTDel 4 solution were also diluted with lOOfg / ⁇ normal sequence control template DNA solution, respectively, to obtain 5 mol% deletion sequence control template DNA solution.
  • the reaction volume is 25 ⁇ 1.
  • 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.
  • 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.
  • Example 2 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.
  • 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 / ⁇ 1 to obtain 1, 0.1, 0.01 mol. % Deficient sequence control template DNA solution was prepared.
  • 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.
  • 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.
  • 0.2 ⁇ F3D3 primer, 0.2 ⁇ F4D4 primer, 0.2 ⁇ M F5D5 primer, 0.2 ⁇ RV primer, 0.2 ⁇ ⁇ probe ⁇ , 0.2 ⁇ probe Prepared to be DF.
  • the reaction volume is 25 ⁇ 1.
  • 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.
  • N is the HEX / FAM value when the normal sequence control template DNA is a cocoon type
  • 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.
  • 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.
  • 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.
  • 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: 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 ⁇ 14 PCR primer F3-2D3 to amplify a deletion mutant "Del-3"
  • SEQ ID N ⁇ 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
  • nucleotides 5 is a ribonucleotides-other nucleoti des are deoxynbonucleotides

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Abstract

La présente invention concerne un procédé permettant de détecter rapidement, de manière commode et avec une haute sensibilité, une mutation dans le gène EGFR responsable d'une déficience; ladite mutation étant impliquée dans la prédiction d'une maladie, de l'effet d'un médicament et d'un effet secondaire. L'invention concerne également un kit permettant cette détection.
PCT/JP2005/023486 2004-12-28 2005-12-21 Procede de detection d'une mutation dans le gene egfr et kit de detection WO2006070667A1 (fr)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO2009129693A1 (fr) * 2008-04-23 2009-10-29 广州益善生物技术有限公司 Sondes, produits à base de puce liquide et procédés pour la détection de mutations du gène egfr
WO2011131145A1 (fr) * 2010-04-23 2011-10-27 广州益善生物技术有限公司 Puce liquide pour la détection de mutations du gène egfr
US11155877B2 (en) * 2007-04-27 2021-10-26 Quest Diagnostics Investments Llc Nucleic acid detection combining amplification with fragmentation

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US11155877B2 (en) * 2007-04-27 2021-10-26 Quest Diagnostics Investments Llc Nucleic acid detection combining amplification with fragmentation
US11965212B2 (en) 2007-04-27 2024-04-23 Quest Diagnostics Investments Llc Nucleic acid detection combining amplification with fragmentation
WO2009129693A1 (fr) * 2008-04-23 2009-10-29 广州益善生物技术有限公司 Sondes, produits à base de puce liquide et procédés pour la détection de mutations du gène egfr
WO2011131145A1 (fr) * 2010-04-23 2011-10-27 广州益善生物技术有限公司 Puce liquide pour la détection de mutations du gène egfr

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