CN102443140A

CN102443140A - Reagent for detecting EGFR gene mutation and application thereof

Info

Publication number: CN102443140A
Application number: CN2010105044239A
Authority: CN
Inventors: 王树; 杨琼; 刘礼兵; 吴尉; 朱春雷; 冯旭利
Original assignee: Institute of Chemistry CAS
Current assignee: Institute of Chemistry CAS
Priority date: 2010-10-09
Filing date: 2010-10-09
Publication date: 2012-05-09
Anticipated expiration: 2030-10-09
Also published as: CN102443140B

Abstract

The invention aims to provide a reagent for detecting EGFR gene mutation and application thereof. The invention provides a compound shown as a formula . The reagent provided by the invention comprises a compound shown in a formula , a compound dGTP-F1 shown in a formula (II), a primer 1 shown in a sequence 1, a primer 2 shown in a sequence 2, a primer 3 shown in a sequence 3, a primer 4 shown in a sequence 4, a primer 5 shown in a sequence 5 and a primer 6 shown in a sequence 6; the EGFR gene mutation means that the 858 th amino acid residue of the EGFR protein is mutated from L to R from the N tail end; the method provided by the invention overcomes various defects of the prior detection technology and has the advantages of high sensitivity, rapidness, visibility, simplicity and convenience and the like. The invention can be applied to the auxiliary analysis of the sensitivity of the cell line and/or the cancer patient to the anticancer drug and guides the doctor to reasonably carry out the anticancer treatment on the patient.

Description

Detect the reagent and the application thereof of EGFR transgenation

Technical field

The present invention relates to a kind of reagent and application thereof of the EGFR of detection transgenation.

Background technology

Detect cancer related gene sudden change quickly and accurately and can important information be provided for clinical cancer molecular diagnosis and treatment.In the non-small cell lung patient, No. 21 exon point mutation of cancerous lung tissue EFGR gene, L858R suddenlys change (being T/G), accounts for 44% of all kinds of sudden changes of EFGR.The detected result of this point mutation can the direct clinical anticancer therapy.Basis and clinical study prove that if point mutation has taken place No. 21 exon point of patients with lung cancer EGFR gene, they are then to anti-lung-cancer medicament, and gefitinib and erlotinib have higher susceptibility.Therefore, this gene mutations detected result has become one of clinically important molecular pathology diagnosis index.

The traditional method that detects transgenation mainly comprises direct dna sequencing method, single-strand conformation polymorphism analysis, Realtime PCR method, restricted enzyme cutting analysis method and mass spectrometric analysis method.These methods have characteristics separately, yet, sensitivity and problem optionally, cost height, complicated operation, cycle are long, and to the drawbacks limit such as particular requirement of instrument their clinical application.Particularly owing to be mixed with normally sudden change tissue in the clinical cancer sample, therefore, the conventional dna sequencing method of using often detects less than transgenation in clinical and the fundamental research.

Summary of the invention

The purpose of this invention is to provide the reagent and the application thereof of a kind of detection EGFR (EGF-R ELISA) transgenation.

Compound shown in the protection of the present invention (I);

formula (I);

The number-average molecular weight of compound is 5000-100000 shown in the formula (I).

The number-average molecular weight of compound specifically can be 3.5 * 10 shown in the formula (I) ⁴

The invention provides a kind of genotypic reagent that is used to detect cell, form by compound shown in the formula (I), compound d GTP-Fl, primer 1, primer 2, primer 3, primer 4, primer 5 and primer 6;

Said compound d GTP-Fl is suc as formula shown in (II);

formula (II);

The nucleotide sequence of said primer 1 is shown in the sequence 1 of sequence table; The nucleotide sequence of said primer 2 is shown in the sequence 2 of sequence table; The nucleotide sequence of said primer 3 is shown in the sequence 3 of sequence table; The nucleotide sequence of said primer 4 is shown in the sequence 4 of sequence table; The nucleotide sequence of said primer 5 is shown in the sequence 5 of sequence table; The nucleotide sequence of said primer 6 is shown in the sequence 6 of sequence table;

The genotype of said cell is that TT is homozygous, GG is homozygous and the TG heterozygous; Said TT is homozygous to be the homozygote (or DNA shown in the sequence 7 of sequence table in the genomic dna is the homozygote of T from 5 ' terminal the 423rd Nucleotide) that is T from 5 ' terminal the 132nd Nucleotide of DNA shown in the sequence 8 of sequence table in the genomic dna; Said GG is homozygous to be the homozygote (or DNA shown in the sequence 7 of sequence table in the genomic dna is the homozygote of G from 5 ' terminal the 423rd Nucleotide) that is G from 5 ' terminal the 132nd Nucleotide of DNA shown in the sequence 8 of sequence table in the genomic dna, and said TC heterozygous is their heterozygote.In the homozygous cell with the TG heterozygous of GG base the proteic L858R sudden change of EGFR having taken place, to anti-lung-cancer medicament gefitinib and erlotinib, has had higher susceptibility.

The present invention also protects a kind of genotypic method that said reagent detects cell of using, and comprises the steps:

(1) genomic dna of extraction cell to be measured;

(2) with the genomic dna be template, the primer of forming with said primer 1 and said primer 2 obtains pcr amplification product to carrying out pcr amplification;

(3) pcr amplification product with step (2) is a template, and the primer of forming with said primer 3 and said primer 4 obtains pcr amplification product to carrying out pcr amplification;

(4) with said compound d GTP-Fl and said primer 5 pcr amplification product of step (3) is carried out compound shown in the adding formula (I) behind the single-basic extension, the exciting light with 380nm carries out fluorescence spectrum scanning then; Detect the fluorescence intensity A1 of 500-540nm and the fluorescence intensity A2 of 400-440nm, the value of A2/A1 is energy transfer ratio T1; With said compound d GTP-Fl and said primer 6 pcr amplification product of step (3) is carried out compound shown in the adding formula (I) behind the single-basic extension, the exciting light with 380nm carries out fluorescence spectrum scanning then; Detect the fluorescence intensity B1 of 500-540nm and the fluorescence intensity B2 of 400-440nm, the value of B2/B1 is energy transfer ratio T2; If T1 greater than 0.3 and T2 less than 0.3, the genotype of cell to be measured is that TT is homozygous; If T1 less than 0.3 and T2 greater than 0.3, the genotype of cell to be measured is that GG is homozygous; If T1 greater than 0.3 and T2 greater than 0.3, the genotype of cell to be measured is the TG heterozygous.

Said cell to be measured can be cancerous cell line; Said cancerous cell line specifically can be A498 clone, NCI-1975 clone, A549 clone, MCF-7 clone, Jurkat-T clone or U251 clone.

The reaction conditions of said single-basic extension specifically can be: 94 ℃ of sex change 3 minutes; 95 ℃ of sex change 1 minute, 57 ℃ were extended 40 circulations 30 seconds; 4 ℃, 10 minutes.

The present invention goes back compound, compound d GTP-Fl, said primer 1, said primer 2, said primer 3, said primer 4, said primer 5 and said primer 6 shown in the protection (I) are used for detecting the reagent of EGFR transgenation in preparation application.

The present invention also protects and a kind ofly uses said reagent and detect the method that whether contains the homozygous or TC heterozygous cell of GG in the biological organization sample to be measured, comprises the steps:

(1) genomic dna of extraction biological organization sample to be measured;

(4) with said compound d GTP-F1 and said primer 5 pcr amplification product of step (3) is carried out compound shown in the adding formula (I) behind the single-basic extension, the exciting light with 380nm carries out fluorescence spectrum scanning then; Detect the fluorescence intensity A1 of 500-540nm and the fluorescence intensity A2 of 400-440nm, the value of A2/A1 * 100% is energy transfer ratio T1; With said compound d GTP-Fl and said primer 6 pcr amplification product of step (3) is carried out compound shown in the adding formula (I) behind the single-basic extension, the exciting light with 380nm carries out fluorescence spectrum scanning then; Detect the fluorescence intensity B1 of 500-540nm and the fluorescence intensity B2 of 400-440nm, the value of B2/B1 is energy transfer ratio T2; If T1 greater than 0.3 and T2 less than 0.3, do not contain the homozygous or TC heterozygous cell of GG in the biological organization sample to be measured; If T1 less than 0.3 and T2 greater than 0.3, or T1 greater than 0.3 and T2 greater than 0.3, contain the homozygous or TC heterozygous cell of GG in the biological organization sample to be measured.

Compare with traditional dna sequencing, it is quick, easy that the present invention handles, hypersensitivity and specificity; Compare with system's (Realtime PCR, MS etc.) of various advanced technology, the present invention need not complicated plant and instrument, and sample preparation is simple, and cost is low.Method provided by the present invention has overcome all deficiencies of existing detection technique, has hypersensitivity, advantage such as quick, visual, easy.The present invention can be applicable to assistant analysis clone with or the cancer patients to the susceptibility of cancer therapy drug, instruct the doctor that patient is rationally carried out anticancer therapy.

Description of drawings

Fig. 1 is the detection principle schematic among the present invention.

Fig. 2 analyzes pcr amplification product figure for agarose gel electrophoresis.

Fig. 3 is different clone energy transfer ratio histograms.

Fig. 4 is the synthesis flow synoptic diagram of compound shown in the formula (I).

Fig. 5 is sensitivity analysis figure as a result; In the electrophorogram, under going up certainly, from left to right, corresponding successively first group to the 17 group of each swimming lane.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Genome extracts test kit and purchases the root biochemical technology company in the sky, and PIN is: DP304.The GoTaq archaeal dna polymerase is purchased the company in Promega, and PIN is M7132.Clone is purchased the preclinical medicine cell centre in preclinical medicine institute of China Concord Medical Science University, is the international standard type; A498 clone (L858R wild type gene homozygote, TT is homozygous) is numbered CCCC0171, the NCI-1975 clone (heterozygote of L858R wild type gene and mutator gene; The TG heterozygous) be numbered CCC0252, A549 clone (L858R wild type gene homozygote, TT is homozygous) is numbered CCC0002; MCF-7 clone (L858R wild type gene homozygote; TT is homozygous) be numbered CCC0013, Jurkat-T clone (L858R wild type gene homozygote, TT is homozygous) is numbered CCC0075; U251 clone (L858R wild type gene homozygote, TT is homozygous) is numbered CCC0058.

Ultimate principle is referring to Fig. 1: in the single base extension system, dna molecular has negative charge, and compound has positive charge shown in the formula (I); Carrying out along with single base extension; Therefore, if dGTP-Fl can be incorporated into 3 ' end of single-basic extension primer, so and compound shown in the dna molecular bonded formula (I) just can and dGTP-Fl between FRET takes place; Thus, can judge whether to have taken place mutator gene; Compound is the water-soluble cationic conjugated polymers shown in the formula (I), and is bigger with the spectra overlapping integration of Fl, and energy transfer preferably can take place, and therefore, not only has characteristics such as remarkable fluorescence amplification, and can improve the specificity that detects.The primer that primer 1 and primer 2 are formed to the target sequence of pcr amplification EGFR gene shown in the sequence 7 of sequence table; The primer of further forming with primer 3 and primer 4 to the target sequence of pcr amplification shown in the sequence 8 of sequence table; ' terminal the 132nd exists a mononucleotide polymorphic T/G (being the L858R sudden change) to DNA shown in the sequence 8, and wild-type is T, and mutant is G from 5; Promptly have three kinds of genotype: TT is homozygous, GG gene pure type and TG heterozygous.

Synthesizing of compound shown in embodiment 1, the formula (I)

9,9-two-(6 '-bromine hexyl)-2,7-dibromo fluorenes: purchase Co. in Synwit Technology, Ltd, the product article No. is: 570414-33-4.

2-isopropoxy-4,4,5,5-tetramethyl--1,3,2-three oxygen borines: purchase the company in Alfa Aesar, the product article No. is 999770-93-1.

The synthesis flow of compound is seen Fig. 4 shown in the formula (I).

1,1,2-two-(4-bromophenyl)-hydrazine synthetic

1. (4.02g 20mmol) packs in the single port bottle of 25ml, adds thionyl chloride (SO with parabromobenzoic acid ₂Cl ₂, 3.63ml 50mmol), drips the pyridine of catalytic amount again, is warming up to 70 ℃, stirs 12h.

2. after 1. step was reacted and stopped, excessive thionyl chloride was removed in air distillation, add the exsiccant N-Methyl pyrrolidone (NMP, 20ml), drip under the room temperature be dissolved with Hydrazine Hydrate 80 (0.5g, dry NMP10ml 10mmol), after dropwising, room temperature reaction 5h.

3. after 2. step is reacted and stopped, pouring reaction solution in the 200ml zero(ppm) water into, stir, generate deposition in a large number, filter collecting precipitation.

4. will precipitate and use wet chemical respectively, zero(ppm) water and ETHYLE ACETATE drip washing, drying under reduced pressure obtains white solid powder 2.7g, productive rate 68%.

¹H-NMR(400MHz，d ⁶-DMSO，ppm)δ：7.74(d，4H，11.3)，7.86(d，4H，11.3)，10.62(s，2H，NH)；ESI-MS(m/z)：398，M+。

Above characterization data explanation, the white solid powder is 1,2-two-(4-bromophenyl)-hydrazine (compound 1 among Fig. 4).

2,1,2-two [chloro-(4-bromophenyl)-methylene radical]-hydrazine synthetic

1. (2.39g is 6mmol) with phosphorus pentachloride (PCl with step 1 synthetic compound 1 ₅, 2.75g 13.2mmol) is dissolved in the 20ml toluene, under nitrogen atmosphere, is warming up to 120 ℃, reaction 3h.

2. after 1. step is reacted and stopped, being cooled to room temperature, removal of solvent under reduced pressure toluene, residuum filter with a large amount of water washings, collect filter residue, obtain bullion after the drying.

3. with the mixed solvent recrystallization of bullion, obtain yellow solid 2.01g, productive rate 77% with ethanol/dichloromethane (volume ratio is 1/1).

The characterization data of yellow solid is following:

1H-NMR(400MHz，d6-DMSO，ppm)δ：7.81(d，4H，8.3)，8.00(d，4H，8.3)。

Above characterization data explanation, yellow solid is 1,2-two [chloro-(4-bromophenyl)-methylene radical]-hydrazine (compound 2 among Fig. 4).

3,3,4,5-triphenyl-1,2,3-triazole synthetic

1. (1.74g, 4mmol) (0.37g 4mmol) is dissolved in 15mlN, and accelerine is warming up to 135 ℃ under nitrogen atmosphere, reaction 12h with aniline with step 2 synthetic compound 2.

2. after 1. step is reacted and stopped, being cooled to room temperature, (in the aqueous hydrochloric acid, stir 30min, filter, collecting precipitation obtains bullion after the drying to pour reaction solution into 30ml l2M.

3. bullion is separated with silica gel column chromatography, developping agent is petrol ether/ethyl acetate (2/1), obtains white solid 1.3g, productive rate 61%.

The characterization data of white solid is following:

¹H-NMR(400MHz，CDCl ₃，ppm)δ：7.01(d，2H，8.5)，7.25(s，2H)，7.28(s，2H)，7.48(d，5H，8.5)，7.60(d，2H，8.5)；ESI-MS(m/z)：453Z，M ⁺。

Above characterization data explanation, white solid is 3,4,5-triphenyl-1,2,3-triazole (compound 3 among Fig. 4).

4,9,9-two-(6 '-bromine hexyl)-2,7-two fluorenes boric acid esters synthetic

1. with 9,9-two-(6 '-bromine hexyl)-2, (2.6g 4mmol) is dissolved in the 50ml anhydrous tetrahydro furan 7-dibromo fluorenes, and temperature is reduced to-78 ℃; (2.95ml 8.4mmol), reacts 15min, adds 2-isopropoxy-4,4 rapidly to add the 2.85M n-Butyl Lithium; 5,5-tetramethyl--1,3,2-three oxygen borine (1.71g; 9.2mmol), stirring 30min, temperature of reaction gets warm again after a cold spell to room temperature gradually, stirs 12h.

2. after 1. step is reacted and stopped, reaction solution is poured in the 50ml water, and with extracted with diethyl ether (30ml * 3 time), merged organic phase, use distilled water wash, anhydrous magnesium sulfate drying filters, and is concentrated, obtains crude product.

3. crude product is used silica gel column chromatography separating purification, developping agent is ethyl acetate/petroleum ether (1/15), gets white solid product 1.84g, productive rate 62%.

The characterization data of white solid is following:

1H-NMR(400MHz，CDCl3，ppm)δ：0.55(m，4H)，1.07(m，4H)，1.14(m，4H)，1.39(s，24H)，1.62(m，4H)，2.03(m，4H)，3.26(m，4H)，7.72(d，4H，J＝7.2)，7.81(d，2H，J＝7.8)。

Above characterization data explanation, white solid is 9,9-two-(6 '-bromine hexyl)-2,7-two fluorenes boric acid esters (compound 4 among Fig. 4).

5, PFN-Br's is synthetic

1. with step 3 synthetic 3,4,5-triphenyl-1; 2, (0.079g is 0.175mmol) with step 4 synthetic 9 for 3-triazole (compound 3); 9-two-(6 '-bromine hexyl)-2, (0.130g 0.175mmol) is dissolved in the 5ml toluene 7-two fluorenes boric acid esters (compound 4); Add 3ml 2M wet chemical, the degassing, nitrogen protection add the palladium catalyst PdCl of catalytic amount down ₂(dppf) (20mg), mixed solution is warming up to 80 ℃ of reaction 2d.

2. after 1. step is reacted and stopped, being cooled to room temperature, toluene decompression is removed, add chloroform, washing twice concentrates organic phase, is added drop-wise in the methyl alcohol and precipitates, and centrifugally obtains thick product.

3. thick product is precipitated twice in methyl alcohol, obtain solid product (37mg, 27%) after the drying.

The characterization data of solid product is following:

1H?NMR(400MHz，CDCl3)：δ＝7.83(m，6H)，7.43-7.51(m，8H)，7.25-7.32(m，5H)，3.30(m，4H)，2.10(b，4H)，1.81(m，4H)，1.39-1.13(m，8H)，0.79(b，4H)。

Above characterization data explanation, solid product is PFN-Br.

With gel permeation chromatography (GPC) method is the number-average molecular weight of standard analysis PFN-Br with the PS, and number-average molecular weight is 3.5 * 10 ⁴

6, PFN-N+ (CH3) 3Br-'s is synthetic

Step 5 synthetic PFN-Br (20mg) is dissolved in the 4ml THF, adds trimethylamine aqueous solution (Trimethylamine 99 that contained), room temperature reaction spends the night, and removes and desolvates and excessive trimethylamine solution, gets product (22mg, 97%) after the drying.

The characterization data of product is following:

1H?NMR(400MHz，DMSO-6)：δ＝7.91-7.79(m，6H)，7.59-7.32(m，13H)，3.18(b，4H)，2.96(b，＞15H)，2.18-1.91(m，4H)，1.47(b，4H)，1.09(b，8H)，0.67(b，4H)。

Above characterization data explanation, solid product is the 3Br-of Compound P FN-N+ (CH3) shown in the formula (I).

Because the 3Br-of Compound P FN-N+ (CH3) shown in the formula (I) compares with PFN-Br, gap only is N, so number-average molecular weight also is 3.5 * 10 ⁴

formula (I).

The composition of embodiment 2, reagent

Reagent is made up of compound shown in the formula (I) of embodiment 1 preparation, compound d GTP-Fl, primer 1, primer 2, primer 3, primer 4, primer 5 and primer 6, and each component is packed separately.

Compound d GTP-F1 is suc as formula shown in (II), and available from Perkin Elmer, catalog number number is: NEL429001EA.

formula (II).

Article six, the sequence of primer is following, and is synthetic by Shanghai biotechnology ltd:

Primer 1:5 '-TCAGCCTGGCAAGTCCAGTAAG-3 ';

Primer 2: 5 '-AGCTCTGGCTCACACTACCAG-3 ';

Primer 3:5 '-CCTCACAGCAGGGTCTTCTC-3 ';

Primer 4:5 '-TGCCTCCTTCTGCATGGTA-3 ';

Primer 5:5 '-AGATCACAGATTTTGGGCT-3 ';

Primer 6:5 '-GATCACAGATTTTGGGCG-3 '.

Primer 1, primer 2, primer 3 and primer 4 are used for through nest-type PRC amplified target gene (EFGR gene), and wherein primer 1 is formed outside primer with primer 2, and primer 3 is formed inboard primer with primer 4.Primer 5 is the T wild-type primers (L858) that are used for single-basic extension.Primer 6 is the G mutant primers (L858R) that are used for single-basic extension.

The reagent of embodiment 3, application implementation example 2 detects A498 clone (cancer cell system)

One, culturing cell system

In containing the DMEM nutrient solution of 10% foetal calf serum, cultivate A498 clone, culture condition is 37 ℃, 5%CO ₂Cell covers with 70% o'clock of culture plate, collecting cell.

Two, the reagent of application implementation example 2 detects A498 clone (cancer cell system)

1, extracts the genomic dna of cell.

2, the genomic dna with step 1 is a template, and the primer of forming with primer 1 and primer 2 is to carrying out pcr amplification, and pcr amplification product carries out the agarose gel electrophoresis (see figure 2), obtains the pcr amplification product of 659bp.

3, the pcr amplification product with step 2 is a template, and the primer of forming with primer 3 and primer 4 is to carrying out pcr amplification, and pcr amplification product carries out the agarose gel electrophoresis (see figure 2), obtains the pcr amplification product of 182bp.

4, carry out single base extension

Reaction system first (10 μ L): contain the pcr amplification product of

100ng step

3,2 μ M compound d GTP-Fl, 1.5U Taq archaeal dna polymerase, 1 μ M primer 5; Buffer system is PCR buffer.

Reaction system second (10 μ L): contain the pcr amplification product of

100ng step

3,2 μ M compound d GTP-Fl, 1.5UTaq archaeal dna polymerase, 1 μ M primer 6; Buffer system is PCR buffer.

The reaction system first is identical with the reaction conditions of reaction system second, is following process: first stage, 94 ℃ of sex change 3 minutes; Second stage, 95 ℃ of sex change 1 minute, 57 ℃ were extended 40 circulations 30 seconds; Three phases, 4 ℃, 10 minutes.

5, fluorescence spectrometry

After step 4 finishes, in the reaction system first, add compound shown in the 15 μ M formulas (I), adopt biotek ELIASA (model is SynergyHT for the U.S., Biotek) to carry out fluorescence spectrum scanning; Excitation wavelength is 380nm, detects the fluorescence intensity (A1) of 500-540nm and the fluorescence intensity (A2) of 400-440nm, calculating energy transfer ratio (A2/A1).The energy transfer ratio is set is 0.3 and be threshold value,, explain and contain wild type gene in the genome if energy shifts Billy greater than 0.3.The energy transfer ratio of reaction system first is 0.71.

After step 4 finishes, in reaction system second, add compound shown in the 15 μ M formulas (I), adopt biotek ELIASA (model is SynergyHT for the U.S., Biotek) to carry out fluorescence spectrum scanning; Excitation wavelength is 380nm, detects the fluorescence intensity (B1) of 500-540nm and the fluorescence intensity (B2) of 400-440nm, calculating energy transfer ratio (B2/B1.The energy transfer ratio is set is 0.3 and be threshold value, contain mutated genes in the genome if the energy transfer ratio greater than 0.3, is explained.The energy transfer ratio of reaction system second is 0.13.

The result shows that A498 clone is that TT is homozygous.

The reagent of embodiment 4, application implementation example 2 detects NCI-1975 clone (cancer cell system)

One, culturing cell system

In containing the DMEM nutrient solution of 10% foetal calf serum, cultivate NCI-1975 clone, culture condition is 37 ℃, 5%CO ₂Cell covers with 70% o'clock of culture plate, collecting cell.

Two, the reagent of application implementation example 2 detects NCI-1975 clone (cancer cell system)

Step 2 with embodiment 3.

The energy transfer ratio of reaction system first is 0.43.

The energy transfer ratio of reaction system second is 0.37.

A498 cell line mcf-7 clone is the TG heterozygous.

The reagent of embodiment 5, application implementation example 2 detects A549 clone (cancer cell system)

One, culturing cell system

In containing the DMEM nutrient solution of 10% foetal calf serum, cultivate A549 clone, culture condition is 37 ℃, 5%CO ₂Cell covers with 70% o'clock of culture plate, collecting cell.

Two, the reagent of application implementation example 2 detects A549 clone (cancer cell system)

Step 2 with embodiment 3.

The energy transfer ratio of reaction system first is 0.71.

The energy transfer ratio of reaction system second is 0.14.

The result shows that A549 clone is that TT is homozygous.

The reagent of embodiment 6, application implementation example 2 detects MCF-7 clone (cancer cell system)

One, culturing cell system

In containing the DMEM nutrient solution of 10% foetal calf serum, cultivate MCF-7 clone, culture condition is 37 ℃, 5%CO ₂Cell covers with 70% o'clock of culture plate, collecting cell.

Two, the reagent of application implementation example 2 detects MCF-7 clone (cancer cell system)

Step 2 with embodiment 3.

The energy transfer ratio of reaction system first is 0.62.

The energy transfer ratio of reaction system second is 0.15.

The result shows that MCF-7 clone is that TT is homozygous.

The reagent of embodiment 7, application implementation example 2 detects Jurkat-T clone (cancer cell system)

One, culturing cell system

In containing the DMEM nutrient solution of 10% foetal calf serum, cultivate Jurkat-T clone, culture condition is 37 ℃, 5%CO ₂Cell covers with 70% o'clock of culture plate, collecting cell.

Two, the reagent of application implementation example 2 detects Jurkat-T clone (cancer cell system)

Step 2 with embodiment 3.

The energy transfer ratio of reaction system first is 0.65.

The energy transfer ratio of reaction system second is 0.14.

The result shows that Jurkat-T clone is that TT is homozygous.

The reagent of embodiment 8, application implementation example 2 detects U251 clone (cancer cell system)

One, culturing cell system

In containing the DMEM nutrient solution of 10% foetal calf serum, cultivate U251 clone, culture condition is 37 ℃, 5%CO ₂Cell covers with 70% o'clock of culture plate, collecting cell.

Two, the reagent of application implementation example 2 detects U251 clone (cancer cell system)

Step 2 with embodiment 3.

The energy transfer ratio of reaction system first is 0.63.

The energy transfer ratio of reaction system second is 0.14.

The result shows that U251 clone is that TT is homozygous.

Among the embodiment 3 to 8, the energy transfer ratio behind each clone single-basic extension is relatively seen table 1 and Fig. 3.

Energy transfer ratio behind each clone single-basic extension of table 1

	A498	NCI-1975	A549	U251	MCF-7	Jurkat-T
							T wild-type primer	0.71	0.43	0.71	0.63	0.62	0.65
G mutant primer	0.13	0.37	0.14	0.14	0.15	0.14

Embodiment 9, sensitivity detect

NCI-1975 clone genomic dna and A549 clone genomic dna are mixed by following mass ratio respectively:

First group: NCI-1975 clone (ratio that is mutator gene is 50%);

Second group: NCI-1975: A549=90: 10 (ratio that is mutator gene is 45%);

The 3rd group: NCI-1975: A549=80: 20 (ratio that is mutator gene is 40%);

The 4th group: NCI-1975: A549=70: 30 (ratio that is mutator gene is 35%);

The 5th group: NCI-1975: A549=60: 40 (ratio that is mutator gene is 30%);

The 6th group: NCI-1975: A549=50: 50 (ratio that is mutator gene is 25%);

The 7th group: NCI-1975: A549=40: 60 (ratio that is mutator gene is 20%);

The 8th group: NCI-1975: A549=32: 68 (ratio that is mutator gene is 16%);

The 9th group: NCI-1975: A549=30: 70 (ratio that is mutator gene is 15%);

The tenth group: NCI-1975: A549=20: 80 (ratio that is mutator gene is 10%);

The 11 group: NCI-1975: A549=16: 84 (ratio that is mutator gene is 8%);

The 12 group: NCI-1975: A549=10: 90 (ratio that is mutator gene is 5%);

The 13 group: NCI-1975: A549=8: 92 (ratio that is mutator gene is 4%);

The 14 group: NCI-1975: A549=4: 96 (ratio that is mutator gene is 2%);

The 15 group: NCI-1975: A549=2: 98 (ratio that is mutator gene is 1%);

The 16 group: NCI-1975: A549=1: 99 (ratio that is mutator gene is 0.5%);

The 17 group: A549 clone (ratio that is mutator gene is 0%).

The reagent of application implementation example 2 detects each group mixture, and method is with the step 2 of embodiment 3.

The result sees Fig. 5.The result shows that the detection sensitivity of present method is 8%, in mixing sample, only contains 8% mutator gene also can be detected by this method if also be.

Claims

1. Compound shown in formula (I);

Formula (I);

The number average molecular weight of the compound represented by formula (I) is 5,000-100,000.

2. The compound according to claim 1, characterized in that the number average molecular weight of the compound represented by formula (I) is 3.5×10 ⁴ .

3. the reagent for detecting the genotype of cell is made up of compound described in claim 1, compound dGTP-F1, primer 1, primer 2, primer 3, primer 4, primer 5 and primer 6;

The compound dGTP-F1 is shown in formula (II);

Formula (II);

The nucleotide sequence of the primer 1 is shown in the sequence 1 of the sequence listing; the nucleotide sequence of the primer 2 is shown in the sequence 2 of the sequence listing; the nucleotide sequence of the primer 3 is shown in the sequence listing 3; the nucleotide sequence of the primer 4 is shown in the sequence 4 of the sequence listing; the nucleotide sequence of the primer 5 is shown in the sequence 5 of the sequence listing; the nucleotide sequence of the primer 6 is shown in the sequence 5 of the sequence listing Shown in sequence 6 of the sequence listing;

The genotypes of the cells are TT homozygous, GG homozygous and TG heterozygous; the TT homozygous is the DNA shown in sequence 8 of the sequence table in the genomic DNA from the 132nd nucleotide at the 5' end It is a homozygote of T, and the GG homozygous type is a homozygous body in which the 132nd nucleotide at the 5' end of the DNA shown in sequence 8 in the genomic DNA sequence table is G, and the TC heterozygous type is their heterozygous fit.

4. The reagent according to claim 3, characterized in that the number average molecular weight of the compound represented by formula (I) is 3.5×10 ⁴ .

5. the method for the genotype of the reagent detection cell of application claim 3 or 4, comprises the steps:

(1) Extracting the genomic DNA of the cells to be tested;

(2) using genomic DNA as a template, performing PCR amplification with a primer pair consisting of the primer 1 and the primer 2 to obtain a PCR amplification product;

(3) using the PCR amplification product of step (2) as a template, performing PCR amplification with a primer pair consisting of the primer 3 and the primer 4, to obtain a PCR amplification product;

(4) Use the compound dGTP-F1 and the primer 5 to carry out single-base extension of the PCR amplification product in step (3) and then add the compound shown in formula (I), then use the excitation light of 380nm to scan the fluorescence spectrum Detect the fluorescence intensity A1 of 500-540nm and the fluorescence intensity A2 of 400-440nm, the value of A2/A1 is energy transfer ratio T1; With described compound dGTP-F1 and described primer 6 the PCR amplification of step (3) Add the compound shown in formula (I) after the amplification product is carried out single base extension, then carry out fluorescence spectrum scanning with the excitation light of 380nm; Detect the fluorescence intensity B1 of 500-540nm and the fluorescence intensity B2 of 400-440nm, the value of B2/B1 It is the energy transfer ratio T2; if T1 is greater than 0.3 and T2 is less than 0.3, the genotype of the cell to be tested is TT homozygous; if T1 is less than 0.3 and T2 is greater than 0.3, the genotype of the cell to be tested is GG homozygous; if If T1 is greater than 0.3 and T2 is greater than 0.3, the genotype of the cells to be tested is TG heterozygous.

6. The method according to claim 5, characterized in that: the cells to be tested are cancer cell lines; the cancer cell lines are specifically A498 cell line, NCI-1975 cell line, A549 cell line, MCF-7 cell line line, Jurkat-T cell line or U251 cell line.

7. The method as claimed in claim 5 or 6, characterized in that: the reaction conditions for the single base extension are: denaturation at 94°C for 3 minutes; denaturation at 95°C for 1 minute, extension at 57°C for 30 seconds, 40 cycles ; 4°C, 10 minutes.

8. the described compound of claim 1 or 2, the compound dGTP-F1 described in claim 3, primer 1, primer 2, primer 3, primer 4, primer 5 and primer 6 are used in the preparation of the genotype of detection cell application in reagents; the genotypes of the cells are TT homozygous, GG homozygous and TG heterozygous; the TT homozygous is DNA shown in sequence 8 of the sequence table in genomic DNA from the 5' end The 132nd nucleotide is a homozygous T, the GG homozygous is the homozygous DNA shown in the sequence 8 of the sequence table in the genomic DNA, the 132nd nucleotide from the 5' end is a G homozygous, and the TC heterozygous The type is their hybrid; the nucleotide sequence of the primer 1 is shown in the sequence 1 of the sequence listing; the nucleotide sequence of the primer 2 is shown in the sequence 2 of the sequence listing; the nucleotide sequence of the primer 3 is The acid sequence is shown in sequence 3 of the sequence listing; the nucleotide sequence of the primer 4 is shown in the sequence 4 of the sequence listing; the nucleotide sequence of the primer 5 is shown in the sequence 5 of the sequence listing; the primer The nucleotide sequence of 6 is shown as sequence 6 in the sequence listing.

9. A method for detecting whether the reagent of claim 3 or 4 contains GG homozygous or TC heterozygous cells in the biological tissue sample to be tested, comprising the steps of:

(1) Extracting the genomic DNA of the biological tissue sample to be tested;

(4) add the compound described in claim 1 or 2 after the PCR amplification product of step (3) is carried out single base extension with described compound dGTP-F1 and described primer 5, then carry out fluorescence spectrum with the excitation light of 380nm Scanning; detect the fluorescence intensity A1 of 500-540nm and the fluorescence intensity A2 of 400-440nm, the value of A2/A1 * 100% is energy transfer ratio T1; Use described compound dGTP-F1 and described primer 6 to step (3 ) of the PCR amplification product is subjected to single base extension and then added to the compound described in claim 1 or 2, and then the excitation light of 380nm is used to scan the fluorescence spectrum; detect the fluorescence intensity B1 of 500-540nm and the fluorescence intensity B2 of 400-440nm, The value of B2/B1 is the energy transfer ratio T2; if T1 is greater than 0.3 and T2 is less than 0.3, the biological tissue sample to be tested does not contain GG homozygous or TC heterozygous cells; if T1 is less than 0.3 and T2 is greater than 0.3, or If T1 is greater than 0.3 and T2 is greater than 0.3, the biological tissue sample to be tested contains GG homozygous cells or TC heterozygous cells.

10. The method according to claim 9, wherein the reaction conditions for the single base extension are: denaturation at 94°C for 3 minutes; denaturation at 95°C for 1 minute, extension at 57°C for 30 seconds, 40 cycles; 4°C ,10 minutes.