CN102251046A - Method for detecting tumor mutant gene in blood - Google Patents
Method for detecting tumor mutant gene in blood Download PDFInfo
- Publication number
- CN102251046A CN102251046A CN2011102185780A CN201110218578A CN102251046A CN 102251046 A CN102251046 A CN 102251046A CN 2011102185780 A CN2011102185780 A CN 2011102185780A CN 201110218578 A CN201110218578 A CN 201110218578A CN 102251046 A CN102251046 A CN 102251046A
- Authority
- CN
- China
- Prior art keywords
- sample
- gene
- damping fluid
- pcr amplification
- volume ratio
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention relates to a method for detecting a tumor mutant gene in blood. The method comprises the following steps of: (1) preparing a polyethylene oxide sieving medium containing TBE (Tetrabromoethane); (2) extracting a genome DNA (Deoxyribonucleic Acid) in a blood sample; (3) amplifying a gene target fragment needing to be detected: performing conventional PCR (Polymerase Chain Reaction) amplification by taking the genome DNA as an amplifying template to obtain a p53 gene PCR amplification sample and a k-ras gene PCR amplification sample; (4) treating the samples: performing denaturation or enzyme digestion on the p53 gene PCR amplification sample and the k-ras gene PCR amplification sample to obtain a denatured sample solution and an enzyme digestion solution respectively; and (5) detecting the samples: adding a 10*SYBR Green I fluorescence dye and the genome DNA into the denatured sample solution and the enzyme digestion solution respectively, uniformly mixing, adding deionized water till a system is 30-50 mu l, automatically filling the polyethylene oxide sieving medium by using a capillary electrophoresis apparatus pressure system and performing CE-SSCP (Capillary Electrophoresis-Single Strand Conformation Polymorphism) detection or CE-RFLP (Capillary Electrophoresis-Restriction Fragment Length Polymorphism) detection respectively. The method is easy to operate, and has high repeatability.
Description
Technical field
The present invention relates to the capillary electrophoresis detection method in the analytical procedure applied research field, relate in particular to a kind of method that is used for detecting blood tumour mutator gene.
Background technology
The detection of transgenation plays crucial effect in clinical disease diagnosis.At present, the method that the examination point mutation usually adopts polyacrylamide gel (PAGE) electrophoresis based on PCR to combine with range gene sudden change detection technique, these methods have certain limitation when satisfying the needs of clinical rapid detection transgenation.Therefore it is imperative to set up a kind of detection method of gene mutation that is used for clinical diagnosis quickly and accurately.
Capillary electrophoresis (CE) is the separate analytical technique that a kind of novel electrophoresis combines with chromatogram, have the separation efficiency height, characteristics such as analysis speed is fast, amount of samples is few, applied range, be widely used in aspects such as molecular biology, molecular medicine and life science.For example SSCP (single strand conformation polymorphism detection), RFLP (restriction fragment length polymorphism) have shown unrivaled superiority in the mutator gene context of detection when combining to CE with range gene sudden change detection technique.Having put down in writing application number in the Chinese patent database in 1984～2010 years is the Chinese patent " a kind of PCR detection method and reagent system of tumour associated gene mutation " of 200510026931.X, the patent No. is 200910042599.4 Chinese patent " being used for gene chip and application thereof that the malignancy individual medications associated gene mutation detects ", application number is the Chinese patent " detecting the method for transgenation " of 200810005523.X, application number is that Chinese patent " rapid detection of the apc gene sudden change " application number of 201010134207.X is 200910201917.7 Chinese patent " a kind of primer and application thereof that is used to detect the K-ras transgenation ", above-mentioned patent only discloses the employing real-time quantitative fluorescence PCR, order-checking, high resolving power solubility curve method, detection method of gene mutation such as fluorescence dicyclo probe technique detect oncogene sudden change in the tissue or in the blood, and adopt capillary electrophoresis in conjunction with SSCP, RFLP is used for detecting the method for blood tumour mutator gene and using and but do not see bibliographical information.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method that is used for detecting blood tumour mutator gene of simple to operate, favorable reproducibility.
For addressing the above problem, a kind of method that is used for detecting blood tumour mutator gene of the present invention may further comprise the steps:
(1) contains the preparation of the polyethylene oxide sieving medium of TBE: polyethylene oxide is added in 1 * tbe buffer liquid, make that final solution concentration is 2.5～3.5%, after the pH value is 7.5～9.0, be stirred to till the solution clarification, this settled solution with 0.22um or 0.45um water system filtering with microporous membrane after, promptly obtain polyethylene oxide sieving medium;
(2) extracting genome DNA in the blood sample: add Tris damping fluid I splitting erythrocyte in blood sample after, supernatant discarded adds Tris damping fluid II and cell mass is suspended and the adding Proteinase K in precipitation, and 2～6h is hatched in 55～57 ℃ of water-baths; Hatch and finish back adding phenol-chloroform protein precipitation, draw supernatant liquor and also in this supernatant liquor, add dehydrated alcohol, obtain deposit D NA; This deposit D NA during to constant weight, promptly gets genomic dna through natural drying at room temperature; The volume ratio of described Tris damping fluid I and described blood sample is 1: 2～3; The volume ratio of described Tris damping fluid II and described blood sample is 1: 0.5～1; The volume ratio of described Proteinase K and described blood sample is 1: 0.03～0.05; The volume ratio of described phenol-chloroform and described blood sample is 1: 1～1.5; The volume ratio of described dehydrated alcohol and described blood sample is 1: 1.5～2.0;
(3) need to detect gene purpose fragment amplification: with described genomic dna as amplification template, adopt pp5 design p53 gene and k-ras gene purpose fragment amplification primer, carry out conventional pcr amplification, promptly get p53 gene PCR amplification sample and k-ras gene PCR amplification sample;
(4) sample preparation: p53 gene PCR amplification sample and k-ras gene PCR are increased sample behind 94～97 ℃ of sex change 8～10min, and ice bath 5～10min obtains the denatured DNA sample solution; Or respectively at p53 gene PCR amplification sample, in k-ras gene PCR amplification sample, add tri-distilled water, Tango damping fluid, and in p53 gene PCR amplification sample, add Msp I restriction endonuclease, in k-ras gene PCR amplification sample, add BstN I restriction endonuclease, hatch 4～8h at 37 ℃, behind 80 ℃ of deactivation 15～20min, obtain enzyme and cut the DNA sample solution; Described p53 gene PCR amplification sample, k-ras gene PCR amplification sample are 1: 1.5～1.8: 0.2～0.3 with the volume ratio of tri-distilled water, Tango damping fluid respectively; The volume ratio of described p53 gene PCR amplification sample and described Msp I restriction endonuclease is 1: 0.1～0.2; The volume ratio of described k-ras gene PCR amplification sample and described BstN I restriction endonuclease is 1: 0.1～0.2;
(5) sample detection:
1. the genomic dna that in 17～22ul denatured DNA sample solution, adds 10 * SYBR Green I fluorescence dye, described step (2) gained respectively, adding deionized water to system behind the mixing is 30～50 μ l; Then, using the capillary electrophoresis apparatus pressure system to fill described polyethylene oxide sieving medium automatically, is that 10～20kv, temperature are to adopt the mode of negative pole-10kv electrokinetic injection to carry out CE-SSCP under 15～20 ℃ the condition to detect and get final product at separation voltage;
2. cut the genomic dna that adds 10 * SYBR Green I fluorescence dye, described step (2) gained in the DNA sample solution respectively at 17～22ul enzyme, adding deionized water to system behind the mixing is 30～50 μ l; Then, using the capillary electrophoresis apparatus pressure system to fill described polyethylene oxide sieving medium automatically, is that 10～20kv, temperature are to adopt the mode of negative pole-10kv electrokinetic injection to carry out CE-RFLP under 15～20 ℃ the condition to detect and get final product at separation voltage.
1 * tbe buffer liquid in the described step (1) is meant adding Tutofusin tris (Tris) 10.8 grams, boric acid 5.5 grams, disodium EDTA (EDTA) 0.74 gram respectively in 800 ml deionized water after stirring and dissolving, adds the damping fluid that deionized water is settled to solution 1 liter of gained.
Blood sample in the described step (2) is meant cancer of the stomach, patients with lung cancer and normal people's peripheral vein blood sample.
Tris damping fluid I in the described step (2) be meant by the pH value be 8.0 and Tris-HCl damping fluid, the Repone K of 10mmol/L, the magnesium chloride of 10mmol/L, the pH value of 10mmol/L be 8.0 and the damping fluid that mixes of the Triton X-100 (Triton X-100) of the EDTA of 2mmol/L and 25mL/L.
Tris damping fluid II in the described step (2) be meant by the pH value be 8.0 and Tris-HCl damping fluid, the Repone K of 10mmol/L, the magnesium chloride of 10mmol/L, the pH value of 10mmol/L be 8.0 and the damping fluid that mixes of EDTA, 0.4mol/L sodium-chlor and the 10g/L sodium lauryl sulphate (SDS) of 2mmol/L.
Phenol-chloroform in the described step (2) is meant the solution of saturated phenol with chloroform equal-volume mixing gained.
Amplification condition in the described step (3) is: 94 ℃ of sex change 30s, and 50～60 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations of concurrence.
Tango damping fluid in the described step (4) is meant by 33mM three (methylol) aminomethane acetate (Tris-acetate), 10mM magnesium acetate (Mg-acetate), 66mM Potassium ethanoate (K-acetate), 0.1mg/ml N, after the two silica-based ethanamides of front three (BSA) of O-mix, add the damping fluid that deionized water is settled to 1 liter of gained; The pH value of described three (methylol) aminomethane acetate (Tris-acetate) is 7.9, and temperature is 37 ℃.
The volume ratio of 10 * SYBR Green I fluorescence dye and described genome DNA sample is 1: 3～1: 4 in the described step (5).
The present invention compared with prior art has the following advantages:
1, the present invention adopts capillary electrophoresis in conjunction with SSCP, RFLP sudden change detection technique, compare with the transgenation of classic flat-plate electrophoresis detection, have good temperature controlling system, effectively controlled temperature is at 15～20 ℃, thereby guaranteed the accuracy of detected result and reappeared new.
2, the present invention as sieving medium, has good separation usefulness with the polyethylene oxide (PEO) that contains 1 * tbe buffer liquid, can finish the detection (referring to Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9) to tumour mutator gene in the blood sample.Simultaneously, selected for use 10 * SYBR green I, effectively improved the sensitivity that detects as fluorescence dye.
3, the present invention is simple to operate, fast and convenient, amount of samples is few, visual result is reliable, favorable reproducibility, can be widely used in the detection of tumour mutator gene in the clinical blood sample.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is the capillary electrophoresis figure of the embodiment of the invention 1.
Fig. 2 is the capillary electrophoresis figure of the embodiment of the invention 2.
Fig. 3 is the capillary electrophoresis figure of the embodiment of the invention 2.
Fig. 4 is the capillary electrophoresis figure of the embodiment of the invention 2.
Fig. 5 is the capillary electrophoresis figure of the embodiment of the invention 2.
Fig. 6 is the capillary electrophoresis figure of the embodiment of the invention 3.
Fig. 7 is the capillary electrophoresis figure of the embodiment of the invention 3.
Fig. 8 is the capillary electrophoresis figure of the embodiment of the invention 3.
Fig. 9 is the capillary electrophoresis figure of the embodiment of the invention 3.
Figure 10 is the capillary electrophoresis figure of the embodiment of the invention 4.
Figure 11 is the capillary electrophoresis figure of the embodiment of the invention 4.
Figure 12 is the capillary electrophoresis figure of the embodiment of the invention 4.
Figure 13 is the capillary electrophoresis figure of the embodiment of the invention 4.
Figure 14 is the capillary electrophoresis figure of the embodiment of the invention 5.
Figure 15 is the capillary electrophoresis figure of the embodiment of the invention 5.
Figure 16 is the capillary electrophoresis figure of the embodiment of the invention 5.
Figure 17 is the capillary electrophoresis figure of the embodiment of the invention 5.
Embodiment
1 one kinds of methods that are used for detecting blood sample tumour mutator gene of embodiment are used for the separation of pUC19 DNA/Msp I/Hpa II DNA Marker.
(1) contain the preparation of the polyethylene oxide sieving medium of TBE:
Polyethylene oxide is added in 1 * tbe buffer liquid, make that final solution concentration is 3.0%, after the pH value is 8.3, and till the DF-101S type magnetic stirring apparatus of producing with Henan Yuhua Instrument Co., Ltd., Gongyi City is stirred to the solution clarification with 400 rev/mins speed, this settled solution promptly obtains polyethylene oxide sieving medium after using 0.45um water system filtering with microporous membrane.
Wherein: 1 * tbe buffer liquid is meant that Tutofusin tris (Tris) 10.8 grams that will add the production of Shanghai bio-engineering corporation in 800 ml deionized water respectively, boric acid 5.5 grams that the living worker in Shanghai company produces, the disodium EDTA (EDTA) 0.74 that the living worker in Shanghai company produces restrain after stirring and dissolving, add the damping fluid that deionized water is settled to solution 1 liter of gained.
The application of this method in separating pUC19 DNA/Msp I/Hpa II DNA Marker may further comprise the steps:
1. use capillary electrophoresis apparatus (P/ACE 5000, U.S. Beckman company), applying pressure is the automatic polyethylene oxide sieving medium that contains TBE of filling of pressure system of 10psi (pound/square inch) when separating.
2. the fluorescence dye (Xiamen hundred letter prestige companies provide) of 10 * SYBR Green I is pressed 4: 1 volume ratio mixing with pUC19 DNA/Msp I/Hpa II DNA Marker (BBI company provides), with-10KV negative pole electrokinetic injection 10s, adding deionized water to system then is 30～50 μ l.
3. be that 15kV, electrophoresis temperature are to carry out electrophoretic separation under 15 ℃ at separation voltage.
Its separation electrophoresis figure obtains 11 chromatographic peaks altogether referring to Fig. 1.1 is 26bp among the figure; 2 is 34bp; 3 is 67bp; 4 is 110 and 111bp; 5 is 147bp; 6 is 190bp; 7 is 242bp; 8 is 331bp; 9 is 404bp; 10 is 489bp; 11 is 501bp.
As can be seen from the figure, this method has been carried out good separation to the different lengths dna fragmentation.
2 one kinds of methods that are used for detecting blood tumour mutator gene of embodiment may further comprise the steps:
(1) contain the preparation of the polyethylene oxide sieving medium of TBE:
Polyethylene oxide is added in 1 * tbe buffer liquid, make that final solution concentration is 2.5%, after pH value is 8.0, be stirred to till the solution clarification, behind this settled solution usefulness 0.22um water system filtering with microporous membrane, promptly obtain polyethylene oxide sieving medium.
Wherein 1 * tbe buffer liquid is with embodiment 1.
(2) extracting genome DNA in the blood sample:
Add Tris damping fluid I splitting erythrocyte in blood sample after, supernatant discarded adds Tris damping fluid II and cell mass is suspended and the adding Proteinase K in precipitation, and 4h is hatched in 55 ℃ of water-baths; Hatch and finish back adding phenol-chloroform protein precipitation, draw supernatant liquor and also in this supernatant liquor, add dehydrated alcohol, ice bath 1h, the centrifugal 2min of 10000rmp obtains deposit D NA; This deposit D NA during to constant weight, promptly gets genomic dna through natural drying at room temperature.
The volume ratio of Tris damping fluid I and blood sample is 1: 2; The volume ratio of Tris damping fluid II and blood sample is 1: 0.5; The volume ratio of Proteinase K and blood sample is 1: 0.03; The volume ratio of phenol-chloroform and blood sample is 1: 1; The volume ratio of dehydrated alcohol and blood sample is 1: 1.5.
Wherein: blood sample is meant patients with gastric cancer peripheral vein blood sample.
Tris damping fluid I be meant by the pH value be 8.0 and Tris-HCl damping fluid, the Repone K of 10mmol/L, the magnesium chloride of 10mmol/L, the pH value of 10mmol/L be 8.0 and the damping fluid that mixes of the Triton X-100 (Triton X-100) of the EDTA of 2mmol/L and 25mL/L.
Tris damping fluid II be meant by the pH value be 8.0 and Tris-HCl damping fluid, the Repone K of 10mmol/L, the magnesium chloride of 10mmol/L, the pH value of 10mmol/L be 8.0 and the damping fluid that mixes of EDTA, 0.4mol/L sodium-chlor and the 10g/L sodium lauryl sulphate (SDS) of 2mmol/L.
Phenol-chloroform is meant the solution of saturated phenol with chloroform equal-volume mixing gained.
(3) need to detect gene purpose fragment amplification: genomic dna as amplification template, is adopted pp5 design p53 gene and k-ras gene purpose fragment amplification primer, carry out conventional pcr amplification, promptly get p53 gene PCR amplification sample and k-ras gene PCR amplification sample.
Wherein amplification condition is: 94 ℃ of sex change 30s, and 50～60 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations of concurrence.
(4) sample preparation: get p53 gene PCR amplification sample and each 22ul of k-ras gene PCR amplification sample respectively, at 96 ℃ of sex change 10min, ice bath 5min obtains the denatured DNA sample solution immediately.
(5) sample detection: add the genomic dna of 10 * SYBR Green I fluorescence dye, step (2) gained in 20ul denatured DNA sample solution respectively, the volume ratio of 10 * SYBR Green I fluorescence dye and genome DNA sample is 1: 3.Adding deionized water to system behind the mixing is 30～50 μ l; Then, using the capillary electrophoresis apparatus pressure system to fill described polyethylene oxide sieving medium automatically, is that 13kv, temperature are to adopt the mode of negative pole-10kv electrokinetic injection to carry out CE-SSCP under 15 ℃ the condition to detect and get final product at separation voltage.
Its separation electrophoresis figure is referring to Fig. 2, Fig. 3, Fig. 4, Fig. 5.Fig. 2, Fig. 3 are respectively p53 gene normal control and tumor sample CE-SSCP detects electrophorogram; Fig. 4, Fig. 5 are respectively k-ras gene normal control and tumor sample CE-SSCP detects electrophorogram.1 is the complete double-stranded DNA of sex change not among the figure, and 2,3 are the normal single stranded DNA of sex change gained, and 4 are the unusual single stranded DNA of sex change gained.
As can be seen from the figure, this is used for blood tumour mutator gene detection method and all can obtains good separation to double-stranded DNA, single stranded DNA.
3 one kinds of methods that are used for detecting blood tumour mutator gene of embodiment may further comprise the steps:
(1) contain the preparation of the polyethylene oxide sieving medium of TBE:
Polyethylene oxide is added in 1 * tbe buffer liquid, make that final solution concentration is 2.5%, after pH value is 7.5, be stirred to till the solution clarification, behind this settled solution usefulness 0.22um water system filtering with microporous membrane, promptly obtain polyethylene oxide sieving medium.
Wherein 1 * tbe buffer liquid is with embodiment 1.
(2) extracting genome DNA in the blood sample:
Add Tris damping fluid I splitting erythrocyte in blood sample after, supernatant discarded adds Tris damping fluid II and cell mass is suspended and the adding Proteinase K in precipitation, and 2h is hatched in 56 ℃ of water-baths; Hatch and finish back adding phenol-chloroform protein precipitation, draw supernatant liquor and also in this supernatant liquor, add dehydrated alcohol, ice bath 1h, the centrifugal 2min of 10000rmp obtains deposit D NA; This deposit D NA during to constant weight, promptly gets genomic dna through natural drying at room temperature.
The volume ratio of Tris damping fluid I and blood sample is 1: 2.5; The volume ratio of Tris damping fluid II and blood sample is 1: 0.55; The volume ratio of Proteinase K and blood sample is 1: 0.04; The volume ratio of phenol-chloroform and blood sample is 1: 1.2; The volume ratio of dehydrated alcohol and blood sample is 1: 1.7.
Wherein: blood sample is meant patients with gastric cancer peripheral vein blood sample.
Tris damping fluid I, Tris damping fluid II, phenol-chloroform are with embodiment 2.
(3) need to detect gene purpose fragment amplification: genomic dna as amplification template, is adopted pp5 design p53 gene and k-ras gene purpose fragment amplification primer, carry out conventional pcr amplification, promptly get p53 gene PCR amplification sample and k-ras gene PCR amplification sample.
Wherein amplification condition is with embodiment 2.
(4) sample preparation: get p53 gene PCR amplification sample and each 22ul of k-ras gene PCR amplification sample respectively, at 94 ℃ of sex change 9min, ice bath 7min obtains the denatured DNA sample solution immediately.
(5) sample detection: add the genomic dna of 10 * SYBR Green I fluorescence dye, step (2) gained in 17ul denatured DNA sample solution respectively, the volume ratio of 10 * SYBR Green I fluorescence dye and genome DNA sample is 1: 3.5.Adding deionized water to system behind the mixing is 30～50 μ l; Then, using the capillary electrophoresis apparatus pressure system to fill described polyethylene oxide sieving medium automatically, is that 10kv, temperature are to adopt the mode of negative pole-10kv electrokinetic injection to carry out CE-SSCP under 20 ℃ the condition to detect and get final product at separation voltage.
Separation electrophoresis figure is referring to Fig. 6, Fig. 7, Fig. 8, Fig. 9.Fig. 6, Fig. 7 are respectively p53 gene normal control and tumor sample CE-SSCP detects electrophorogram; Fig. 8, Fig. 9 are respectively k-ras gene normal control and tumor sample CE-SSCP detects electrophorogram.1 is the complete double-stranded DNA of sex change not among the figure, and 2,3 are the normal single stranded DNA of sex change gained, and 4,5 are the unusual single stranded DNA of sex change gained.Wherein k-ras gene generation disappearance only detects a strand among Fig. 9.
As can be seen from the figure, this is used for blood tumour mutator gene detection method and all can obtains good separation to double-stranded DNA, single stranded DNA.
4 one kinds of methods that are used for detecting blood tumour mutator gene of embodiment may further comprise the steps:
(1) contain the preparation of the polyethylene oxide sieving medium of TBE:
Polyethylene oxide is added in 1 * tbe buffer liquid, make that final solution concentration is 2.5%, after pH value is 8.5, be stirred to till the solution clarification, behind this settled solution usefulness 0.35um water system filtering with microporous membrane, promptly obtain polyethylene oxide sieving medium.
Wherein 1 * tbe buffer liquid is with embodiment 1.
(2) extracting genome DNA in the blood sample:
Add Tris damping fluid I splitting erythrocyte in blood sample after, supernatant discarded adds Tris damping fluid II and cell mass is suspended and the adding Proteinase K in precipitation, and 4h is hatched in 55 ℃ of water-baths; Hatch and finish back adding phenol-chloroform protein precipitation, draw supernatant liquor and also in this supernatant liquor, add dehydrated alcohol, ice bath 1h, the centrifugal 2min of 10000rmp obtains deposit D NA; This deposit D NA during to constant weight, promptly gets genomic dna through natural drying at room temperature.
The volume ratio of Tris damping fluid I and blood sample is 1: 2; The volume ratio of Tris damping fluid II and blood sample is 1: 0.5; The volume ratio of Proteinase K and blood sample is 1: 0.03; The volume ratio of phenol-chloroform and blood sample is 1: 1; The volume ratio of dehydrated alcohol and blood sample is 1: 1.5.
Wherein: blood sample is meant patients with gastric cancer peripheral vein blood sample.
Tris damping fluid I, Tris damping fluid II, phenol-chloroform are with embodiment 2.
(3) need to detect gene purpose fragment amplification: genomic dna as amplification template, is adopted pp5 design p53 gene and k-ras gene purpose fragment amplification primer, carry out conventional pcr amplification, promptly get p53 gene PCR amplification sample and k-ras gene PCR amplification sample.
Wherein amplification condition is with embodiment 2.
(4) sample preparation: get p53 gene PCR amplification sample and each 22ul of k-ras gene PCR amplification sample respectively, at 97 ℃ of sex change 8min, ice bath 10min obtains the denatured DNA sample solution immediately.
(5) sample detection: add the genomic dna of 10 * SYBR Green I fluorescence dye, step (2) gained in 22ul denatured DNA sample solution respectively, the volume ratio of 10 * SYBR Green I fluorescence dye and genome DNA sample is 1: 3.Adding deionized water to system behind the mixing is 30～50 μ l; Then, using the capillary electrophoresis apparatus pressure system to fill described polyethylene oxide sieving medium automatically, is that 20kv, temperature are to adopt the mode of negative pole-10kv electrokinetic injection to carry out CE-SSCP under 18 ℃ the condition to detect and get final product at separation voltage.
Its separation electrophoresis figure is referring to Figure 10, Figure 11, Figure 12, Figure 13.Figure 10, Figure 11 are respectively p53 gene normal control and tumor sample CE-RFLP detects electrophorogram; Figure 12, Figure 13 are respectively k-ras gene normal control and tumor sample CE-RFLP detects electrophorogram.Wherein Figure 10 cuts gained 53bp and 147bp two bar segment through enzyme, and the single fragment of 200bp that enzyme cuts gained is failed in the visible because sudden change of Figure 11.Wherein Figure 12 cuts gained 45bp and 117bp two bar segment through enzyme, and the single fragment of 162bp that enzyme cuts gained is failed in the visible because sudden change of Figure 13.
As can be seen from the figure, this is used for that blood tumour mutator gene detection method is cut tumour to enzyme and the normal control sample all can obtain good separation.
5 one kinds of methods that are used for detecting blood tumour mutator gene of embodiment may further comprise the steps:
(1) contain the preparation of the polyethylene oxide sieving medium of TBE:
Polyethylene oxide is added in 1 * tbe buffer liquid, make that final solution concentration is 3.5%, after pH value is 9.0, be stirred to till the solution clarification, behind this settled solution usefulness 0.45um water system filtering with microporous membrane, promptly obtain polyethylene oxide sieving medium.
Wherein 1 * tbe buffer liquid is with embodiment 1.
(2) extracting genome DNA in the blood sample:
Add Tris damping fluid I splitting erythrocyte in blood sample after, supernatant discarded adds Tris damping fluid II and cell mass is suspended and the adding Proteinase K in precipitation, and 6h is hatched in 57 ℃ of water-baths; Hatch and finish back adding phenol-chloroform protein precipitation, draw supernatant liquor and also in this supernatant liquor, add dehydrated alcohol, ice bath 1h, the centrifugal 2min of 10000rmp obtains deposit D NA; This deposit D NA during to constant weight, promptly gets genomic dna through natural drying at room temperature.
The volume ratio of Tris damping fluid I and blood sample is 1: 3; The volume ratio of Tris damping fluid II and blood sample is 1: 1; The volume ratio of Proteinase K and blood sample is 1: 0.05; The volume ratio of phenol-chloroform and blood sample is 1: 1.5; The volume ratio of dehydrated alcohol and blood sample is 1: 2.0.
Wherein: blood sample is meant patients with lung cancer peripheral vein blood sample.
Tris damping fluid I, Tris damping fluid II, phenol-chloroform are with embodiment 2.
(3) need to detect gene purpose fragment amplification: genomic dna as amplification template, is adopted pp5 design p53 gene and k-ras gene purpose fragment amplification primer, carry out conventional pcr amplification, promptly get p53 gene PCR amplification sample and k-ras gene PCR amplification sample.
Wherein amplification condition is with embodiment 2.
(4) sample preparation: respectively at p53 gene PCR amplification sample, in k-ras gene PCR amplification sample, add tri-distilled water, Tango damping fluid, and in p53 gene PCR amplification sample, add Msp I restriction endonuclease, in k-ras gene PCR amplification sample, add BstN I restriction endonuclease, hatch 4～8h at 37 ℃, behind 80 ℃ of deactivation 15～20min, obtain enzyme and cut the DNA sample solution.
Wherein: the Tango damping fluid is meant by 33mM three (methylol) aminomethane acetate (Tris-acetate), 10mM magnesium acetate (Mg-acetate), 66mM Potassium ethanoate (K-acetate), 0.1mg/ml N, after the two silica-based ethanamides of front three (BSA) of O-mix, add the damping fluid that deionized water is settled to 1 liter of gained.The pH value of three (methylol) aminomethane acetate (Tris-acetate) is 7.9, and temperature is 37 ℃.
P53 gene PCR amplification sample, k-ras gene PCR amplification sample are 1: 1.8: 0.3 with the volume ratio of tri-distilled water, Tango damping fluid respectively; The volume ratio of p53 gene PCR amplification sample and Msp I restriction endonuclease is 1: 0.2; The volume ratio of k-ras gene PCR amplification sample and BstN I restriction endonuclease is 1: 0.2.
(5) sample detection: cut the genomic dna that adds 10 * SYBR Green I fluorescence dye, step (2) gained in the DNA sample solution respectively at the 22ul enzyme, the volume ratio of 10 * SYBR Green I fluorescence dye and genome DNA sample is 1: 4.Adding deionized water to system behind the mixing is 30～50 μ l; Then, using the capillary electrophoresis apparatus pressure system to fill described polyethylene oxide sieving medium automatically, is that 15kv, temperature are to adopt the mode of negative pole-10kv electrokinetic injection to carry out CE-RFLP under 18 ℃ the condition to detect and get final product at separation voltage.
Its separation electrophoresis figure is referring to Figure 14, Figure 15, Figure 16, Figure 17.Figure 14, Figure 15 are respectively p53 gene normal control and tumor sample CE-RFLP detects electrophorogram; Figure 16, Figure 17 are respectively k-ras gene normal control and tumor sample CE-RFLP detects electrophorogram.Wherein Figure 14 cuts gained 87bp and 94bp two bar segment through enzyme, and the single fragment of 181bp that enzyme cuts gained is failed in the visible because sudden change of Figure 15.Wherein Figure 16 cuts gained 39bp and 136bp two bar segment through enzyme, and the single fragment of 175bp that enzyme cuts gained is failed in the visible because sudden change of Figure 17.
As can be seen from the figure, this is used for that blood tumour mutator gene detection method is cut tumour to enzyme and the normal control sample all can obtain good separation.
6 one kinds of methods that are used for detecting blood tumour mutator gene of embodiment may further comprise the steps:
(1) contain the preparation of the polyethylene oxide sieving medium of TBE:
Polyethylene oxide is added in 1 * tbe buffer liquid, make that final solution concentration is 3.5%, after pH value is 9.0, be stirred to till the solution clarification, behind this settled solution usefulness 0.45um water system filtering with microporous membrane, promptly obtain polyethylene oxide sieving medium.
Wherein 1 * tbe buffer liquid is with embodiment 1.
(2) extracting genome DNA in the blood sample:
Add Tris damping fluid I splitting erythrocyte in blood sample after, supernatant discarded adds Tris damping fluid II and cell mass is suspended and the adding Proteinase K in precipitation, and 6h is hatched in 57 ℃ of water-baths; Hatch and finish back adding phenol-chloroform protein precipitation, draw supernatant liquor and also in this supernatant liquor, add dehydrated alcohol, ice bath 1h, the centrifugal 2min of 10000rmp obtains deposit D NA; This deposit D NA during to constant weight, promptly gets genomic dna through natural drying at room temperature.
The volume ratio of Tris damping fluid I and blood sample is 1: 3; The volume ratio of Tris damping fluid II and blood sample is 1: 1; The volume ratio of Proteinase K and blood sample is 1: 0.05; The volume ratio of phenol-chloroform and blood sample is 1: 1.5; The volume ratio of dehydrated alcohol and blood sample is 1: 2.0.
Wherein: blood sample is meant patients with lung cancer peripheral vein blood sample.
Tris damping fluid I, Tris damping fluid II, phenol-chloroform are with embodiment 2.
(3) need to detect gene purpose fragment amplification: genomic dna as amplification template, is adopted pp5 design p53 gene and k-ras gene purpose fragment amplification primer, carry out conventional pcr amplification, promptly get p53 gene PCR amplification sample and k-ras gene PCR amplification sample.
Wherein amplification condition is with embodiment 2.
(4) sample preparation: respectively at p53 gene PCR amplification sample, in k-ras gene PCR amplification sample, add tri-distilled water, Tango damping fluid, and in p53 gene PCR amplification sample, add Msp I restriction endonuclease, in k-ras gene PCR amplification sample, add BstN I restriction endonuclease, hatch 4～8h at 37 ℃, behind 80 ℃ of deactivation 15～20min, obtain enzyme and cut the DNA sample solution.
Wherein: the Tango damping fluid is with embodiment 5.
P53 gene PCR amplification sample, k-ras gene PCR amplification sample are 1: 1.5: 0.2 with the volume ratio of tri-distilled water, Tango damping fluid respectively; The volume ratio of p53 gene PCR amplification sample and Msp I restriction endonuclease is 1: 0.1; The volume ratio of k-ras gene PCR amplification sample and BstN I restriction endonuclease is 1: 0.1.
(5) sample detection: cut the genomic dna that adds 10 * SYBR Green I fluorescence dye, step (2) gained in the DNA sample solution respectively at the 17ul enzyme, 10 * SYBR Green I fluorescence dye is 1: 3 with the volume ratio of stating genome DNA sample.Adding deionized water to system behind the mixing is 30～50 μ l; Then, using the capillary electrophoresis apparatus pressure system to fill described polyethylene oxide sieving medium automatically, is that 10kv, temperature are to adopt the mode of negative pole-10kv electrokinetic injection to carry out CE-RFLP under 20 ℃ the condition to detect and get final product at separation voltage.
7 one kinds of methods that are used for detecting blood tumour mutator gene of embodiment may further comprise the steps:
(1) contain the preparation of the polyethylene oxide sieving medium of TBE:
Polyethylene oxide is added in 1 * tbe buffer liquid, make that final solution concentration is 3.5%, after pH value is 9.0, be stirred to till the solution clarification, behind this settled solution usefulness 0.45um water system filtering with microporous membrane, promptly obtain polyethylene oxide sieving medium.
Wherein 1 * tbe buffer liquid is with embodiment 1.
(2) extracting genome DNA in the blood sample:
Add Tris damping fluid I splitting erythrocyte in blood sample after, supernatant discarded adds Tris damping fluid II and cell mass is suspended and the adding Proteinase K in precipitation, and 6h is hatched in 57 ℃ of water-baths; Hatch and finish back adding phenol-chloroform protein precipitation, draw supernatant liquor and also in this supernatant liquor, add dehydrated alcohol, ice bath 1h, the centrifugal 2min of 10000rmp obtains deposit D NA; This deposit D NA during to constant weight, promptly gets genomic dna through natural drying at room temperature.
The volume ratio of Tris damping fluid I and blood sample is 1: 3; The volume ratio of Tris damping fluid II and blood sample is 1: 1; The volume ratio of Proteinase K and blood sample is 1: 0.05; The volume ratio of phenol-chloroform and blood sample is 1: 1.5; The volume ratio of dehydrated alcohol and blood sample is 1: 2.0.
Wherein: blood sample is meant patients with lung cancer peripheral vein blood sample.
Tris damping fluid I, Tris damping fluid II, phenol-chloroform are with embodiment 2.
(3) need to detect gene purpose fragment amplification: genomic dna as amplification template, is adopted pp5 design p53 gene and k-ras gene purpose fragment amplification primer, carry out conventional pcr amplification, promptly get p53 gene PCR amplification sample and k-ras gene PCR amplification sample.
Wherein amplification condition is with embodiment 2.
(4) sample preparation: respectively at p53 gene PCR amplification sample, in k-ras gene PCR amplification sample, add tri-distilled water, Tango damping fluid, and in p53 gene PCR amplification sample, add Msp I restriction endonuclease, in k-ras gene PCR amplification sample, add BstN I restriction endonuclease, hatch 4～8h at 37 ℃, behind 80 ℃ of deactivation 15～20min, obtain enzyme and cut the DNA sample solution.
Wherein: the Tango damping fluid is with embodiment 5.
P53 gene PCR amplification sample, k-ras gene PCR amplification sample are 1: 1.6: 0.25 with the volume ratio of tri-distilled water, Tango damping fluid respectively; The volume ratio of p53 gene PCR amplification sample and Msp I restriction endonuclease is 1: 0.15; The volume ratio of k-ras gene PCR amplification sample and BstN I restriction endonuclease is 1: 0.15.
(5) sample detection: cut the genomic dna that adds 10 * SYBR Green I fluorescence dye, step (2) gained in the DNA sample solution respectively at the 20ul enzyme, the volume ratio of 10 * SYBR Green I fluorescence dye and described genome DNA sample is 1: 3.5.Adding deionized water to system behind the mixing is 30～50 μ l; Then, using the capillary electrophoresis apparatus pressure system to fill described polyethylene oxide sieving medium automatically, is that 20kv, temperature are to adopt the mode of negative pole-10kv electrokinetic injection to carry out CE-RFLP under 15 ℃ the condition to detect and get final product at separation voltage.
Claims (9)
1. method that is used for detecting blood tumour mutator gene may further comprise the steps:
(1) contains the preparation of the polyethylene oxide sieving medium of TBE: polyethylene oxide is added in 1 * tbe buffer liquid, make that final solution concentration is 2.5～3.5%, after the pH value is 7.5～9.0, be stirred to till the solution clarification, this settled solution with 0.22um or 0.45um water system filtering with microporous membrane after, promptly obtain polyethylene oxide sieving medium;
(2) extracting genome DNA in the blood sample: add Tris damping fluid I splitting erythrocyte in blood sample after, supernatant discarded adds Tris damping fluid II and cell mass is suspended and the adding Proteinase K in precipitation, and 2～6h is hatched in 55～57 ℃ of water-baths; Hatch and finish back adding phenol-chloroform protein precipitation, draw supernatant liquor and also in this supernatant liquor, add dehydrated alcohol, obtain deposit D NA; This deposit D NA during to constant weight, promptly gets genomic dna through natural drying at room temperature; The volume ratio of described Tris damping fluid I and described blood sample is 1: 2～3; The volume ratio of described Tris damping fluid II and described blood sample is 1: 0.5～1; The volume ratio of described Proteinase K and described blood sample is 1: 0.03～0.05; The volume ratio of described phenol-chloroform and described blood sample is 1: 1～1.5; The volume ratio of described dehydrated alcohol and described blood sample is 1: 1.5～2.0;
(3) need to detect gene purpose fragment amplification: with described genomic dna as amplification template, adopt pp5 design p53 gene and k-ras gene purpose fragment amplification primer, carry out conventional pcr amplification, promptly get p53 gene PCR amplification sample and k-ras gene PCR amplification sample;
(4) sample preparation: p53 gene PCR amplification sample and k-ras gene PCR are increased sample behind 94～97 ℃ of sex change 8～10min, and ice bath 5～10min obtains the denatured DNA sample solution; Or respectively at p53 gene PCR amplification sample, in k-ras gene PCR amplification sample, add tri-distilled water, Tango damping fluid, and in p53 gene PCR amplification sample, add Msp I restriction endonuclease, in k-ras gene PCR amplification sample, add BstN I restriction endonuclease, hatch 4～8h at 37 ℃, behind 80 ℃ of deactivation 15～20min, obtain enzyme and cut the DNA sample solution; Described p53 gene PCR amplification sample, k-ras gene PCR amplification sample are 1: 1.5～1.8: 0.2～0.3 with the volume ratio of tri-distilled water, Tango damping fluid respectively; The volume ratio of described p53 gene PCR amplification sample and described Msp I restriction endonuclease is 1: 0.1～0.2; The volume ratio of described k-ras gene PCR amplification sample and described BstN I restriction endonuclease is 1: 0.1～0.2;
(5) sample detection:
1. the genomic dna that in 17～22ul denatured DNA sample solution, adds 10 * SYBR Green I fluorescence dye, described step (2) gained respectively, adding deionized water to system behind the mixing is 30～50 μ l; Then, using the capillary electrophoresis apparatus pressure system to fill described polyethylene oxide sieving medium automatically, is that 10～20kv, temperature are to adopt the mode of negative pole-10kv electrokinetic injection to carry out CE-SSCP under 15～20 ℃ the condition to detect and get final product at separation voltage;
2. cut the genomic dna that adds 10 * SYBR Green I fluorescence dye, described step (2) gained in the DNA sample solution respectively at 17～22ul enzyme, adding deionized water to system behind the mixing is 30～50 μ 1; Then, using the capillary electrophoresis apparatus pressure system to fill described polyethylene oxide sieving medium automatically, is that 10～20kv, temperature are to adopt the mode of negative pole-10kv electrokinetic injection to carry out CE-RFLP under 15～20 ℃ the condition to detect and get final product at separation voltage.
2. a kind of method that is used for detecting blood tumour mutator gene as claimed in claim 1, it is characterized in that: the 1 * tbe buffer liquid in the described step (1) is meant adding Tutofusin tris 10.8 grams, boric acid 5.5 grams, disodium EDTA 0.74 gram respectively in 800 ml deionized water after stirring and dissolving, adds the damping fluid that deionized water is settled to solution 1 liter of gained.
3. a kind of method that is used for detecting blood tumour mutator gene as claimed in claim 1 is characterized in that: the blood sample in the described step (2) is meant cancer of the stomach, patients with lung cancer and normal people's peripheral vein blood sample.
4. a kind of method that is used for detecting blood tumour mutator gene as claimed in claim 1 is characterized in that: the Tris damping fluid I in the described step (2) be meant by the pH value be 8.0 and Tris-HCl damping fluid, the Repone K of 10mmol/L, the magnesium chloride of 10mmol/L, the pH value of 10mmol/L be 8.0 and the damping fluid that mixes of the Triton X-100 of the EDTA of 2mmol/L and 25mL/L.
5. a kind of method that is used for detecting blood tumour mutator gene as claimed in claim 1 is characterized in that: the Tris damping fluid II in the described step (2) be meant by the pH value be 8.0 and Tris-HCl damping fluid, the Repone K of 10mmol/L, the magnesium chloride of 10mmol/L, the pH value of 10mmol/L be 8.0 and the damping fluid that mixes of EDTA, 0.4mol/L sodium-chlor and the 10g/L sodium lauryl sulphate of 2mmol/L.
6. a kind of method that is used for detecting blood tumour mutator gene as claimed in claim 1 is characterized in that: the phenol-chloroform in the described step (2) is meant the solution of saturated phenol with chloroform equal-volume mixing gained.
7. a kind of method that is used for detecting blood tumour mutator gene as claimed in claim 1 is characterized in that: the amplification condition in the described step (3) is: 94 ℃ of sex change 30s, and 50～60 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations of concurrence.
8. a kind of method that is used for detecting blood tumour mutator gene as claimed in claim 1, it is characterized in that: the Tango damping fluid in the described step (4) is meant by 33mM three (methylol) aminomethane acetate, 10mM magnesium acetate, 66mM Potassium ethanoate, 0.1mg/ml N, after the silica-based ethanamide of the two front threes of O-mixes, add the damping fluid that deionized water is settled to 1 liter of gained; The pH value of described three (methylol) aminomethane acetate is 7.9, and temperature is 37 ℃.
9. a kind of method that is used for detecting blood tumour mutator gene as claimed in claim 1 is characterized in that: the volume ratio of 10 * SYBR Green I fluorescence dye and described genome DNA sample is 1: 3～1: 4 in the described step (5).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102185780A CN102251046A (en) | 2011-07-29 | 2011-07-29 | Method for detecting tumor mutant gene in blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102185780A CN102251046A (en) | 2011-07-29 | 2011-07-29 | Method for detecting tumor mutant gene in blood |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102251046A true CN102251046A (en) | 2011-11-23 |
Family
ID=44978614
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011102185780A Pending CN102251046A (en) | 2011-07-29 | 2011-07-29 | Method for detecting tumor mutant gene in blood |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102251046A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107677823A (en) * | 2016-08-02 | 2018-02-09 | 北京金沐医疗科技有限公司 | A kind of protein chip kit and its detection method for being used to detect tumour antigen |
| CN107677820A (en) * | 2016-08-02 | 2018-02-09 | 北京金沐医疗科技有限公司 | A kind of kit and its detection method for detecting tumour antigen |
| CN110387419A (en) * | 2019-08-20 | 2019-10-29 | 裕策医疗器械江苏有限公司 | Solid tumor polygenes detects genetic chip and preparation method thereof and detection device |
| WO2021190124A1 (en) * | 2020-03-23 | 2021-09-30 | 南京梅傲红清生物科技有限公司 | Application of red blood cell nucleic acid in identifying tumor mutation types |
-
2011
- 2011-07-29 CN CN2011102185780A patent/CN102251046A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107677823A (en) * | 2016-08-02 | 2018-02-09 | 北京金沐医疗科技有限公司 | A kind of protein chip kit and its detection method for being used to detect tumour antigen |
| CN107677820A (en) * | 2016-08-02 | 2018-02-09 | 北京金沐医疗科技有限公司 | A kind of kit and its detection method for detecting tumour antigen |
| CN110387419A (en) * | 2019-08-20 | 2019-10-29 | 裕策医疗器械江苏有限公司 | Solid tumor polygenes detects genetic chip and preparation method thereof and detection device |
| CN110387419B (en) * | 2019-08-20 | 2023-06-13 | 裕策医疗器械江苏有限公司 | Gene chip for detecting multiple genes of entity rumen, preparation method and detection device thereof |
| WO2021190124A1 (en) * | 2020-03-23 | 2021-09-30 | 南京梅傲红清生物科技有限公司 | Application of red blood cell nucleic acid in identifying tumor mutation types |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109468384B (en) | Composite amplification detection kit for simultaneously detecting 45Y loci | |
| CN111560482A (en) | Detection method based on CRISPR/Cas and nucleic acid test paper and human papilloma virus detection kit | |
| CN102618624A (en) | Kit for screening deaf gene of Chinese populations, and use method thereof | |
| CN104024426B (en) | The method for tracing of testing sample and detection kit in the secondary sequencing technologies of DNA | |
| CN105296619A (en) | Kit for SNP typing of obesity-prone genes of Chinese population and using method of kit | |
| CN103820559B (en) | The composite amplification system of 21 STR and test kit | |
| CN106350590A (en) | DNA library construction method for high-throughput sequencing | |
| CN102251046A (en) | Method for detecting tumor mutant gene in blood | |
| CN109517881A (en) | A kind of high-throughput sequencing library construction method of body fluid micro free RNA | |
| CN107513578A (en) | A kind of nucleic acid Mass Spectrometry detection method early sieved for lung cancer driving gene and tumor susceptibility gene | |
| CN109593847B (en) | Primer pair, kit and method for detecting stability of NR24 locus of microsatellite | |
| CN107603971B (en) | Preparation method of in-situ hybridization probe | |
| US10323241B2 (en) | Method for recovering short-chain nucleic acids | |
| CN106497916A (en) | A kind of construction method in the NK cell polygenic variations library for high-flux sequence detection and its application | |
| CN105132571A (en) | Method for detecting free mtDNA (Mitochondrial Deoxyribonucleic Acid) content of peripheral blood by utilizing micro-droplet digital PCR (Polymerase Chain Reaction) detection | |
| CN110791813A (en) | Method for processing single-stranded DNA and application | |
| CN107641649B (en) | Primer pair, kit and method for detecting stability of NR27 locus of microsatellite | |
| CN105238861A (en) | Kit for Chinese pediatric asthma susceptibility gene SNP (single nucleotide polymorphism) genotyping and application method of kit for Chinese pediatric asthma susceptibility gene SNP genotyping | |
| CN103757109B (en) | Primer composition and multiple-gene detection kit for guiding administration of thiazine diuresis drugs and application method thereof | |
| CN108913774B (en) | C-KIT somatic mutation gene detection KIT and detection method thereof | |
| CN109266653B (en) | Reagent, device and method for capturing drug-resistant heterogeneous circulating tumor cells and performing gene analysis | |
| CN107988334B (en) | Method for SNP typing by direct PCR of oral swab | |
| CN103571959B (en) | Primer, probe and kit for detecting gene locus mutation, and using methods | |
| CN105821145A (en) | Primer and method for detecting HLA-DQ gene rs9275319 site polymorphism | |
| CN103131782B (en) | Kit for detecting early stage non-small-cell lung cancer multi-site association genes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20111123 |