CN101550447A - Method for detecting sperm DNA fragments by using terminal transferase mediated biotin labeling dispersed chromatin - Google Patents
Method for detecting sperm DNA fragments by using terminal transferase mediated biotin labeling dispersed chromatin Download PDFInfo
- Publication number
- CN101550447A CN101550447A CNA2009100149335A CN200910014933A CN101550447A CN 101550447 A CN101550447 A CN 101550447A CN A2009100149335 A CNA2009100149335 A CN A2009100149335A CN 200910014933 A CN200910014933 A CN 200910014933A CN 101550447 A CN101550447 A CN 101550447A
- Authority
- CN
- China
- Prior art keywords
- sperm
- gram
- slide glass
- liquid
- milliliters
- 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.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting sperm DNA fragments by using terminal transferase mediated biotin labeling dispersed chromatin, which comprises the following steps: a glass slide carrying with a processed sperm sample is cold-stored at an ambient temperature; the glass slide after cold storage is taken out and immersed in hydrochloric acid at the room temperature so as to melt the DNA of sperm; the glass slide after treatment is immersed into sperm lysate and then put into a buffer solution; the glass slide is taken out from the buffer solution and undergoes dehydration with ethanol; and then a terminal transferase mediated biotin labeling reaction mixture is prepared; the glass slide after treatment is put into a phosphoric acid buffer solution for being washed for three times, and then the terminal transferase mediated biotin labeling reaction mixture is fetched and put on the glass slide; a working solution is dropped on the glass slide after reaction for a reaction without light; and then the glass slide is washed with distilled water; and sperm staining solution A and B are dropped on the glass slide under a microscope and is observed after being dried. The method does not require expensive detecting instruments and reagents; the sperm sample can be stored; detecting result is reliable, with clear halo ring and high resolution ratio.
Description
Technical field
The present invention relates to the cellular elements thing and learn, is a kind of method that detects DNA fragment with spermatozoon with terminal transferase mediated biotin labeling dispersed chromatin.
Background technology
Because the integrity of sperm DNA whether, directly influences the male fertility quality, therefore, this area disclosed the method for multiple detection sperm integrity in recent years, was intended to be used for clinical, improved the accuracy of diagnosis male infertility, and effectively avoided the generation of bad heredity.Yet, these methods are found all to exist big not enough in application: for example: methods such as acridine orange dyeing, comet experiment and fluorescence TUNEL, need to use expensive instrument analyses such as flow cytometer or fluorescent microscope, and reagent costs an arm and a leg, make clinical application be subjected to bigger restriction, the sperm DNA detection method can't be applied; Simultaneously, the detection sample that uses in some method be difficult for to be preserved, and also some method is lower etc. to the quality judgment accuracy of sperm.
Summary of the invention
The purpose of this invention is to provide and a kind ofly detect the method for DNA fragment with spermatozoon with terminal transferase mediated biotin labeling dispersed chromatin, it does not need, and expensive detecting instrument and reagent, sperm sample can be preserved, test-results is reliable, halo is clear, resolving power is high.
The present invention is achieved through the following technical solutions for achieving the above object: a kind of method with terminal transferase mediated biotin labeling dispersed chromatin detection DNA fragment with spermatozoon, and its step is as follows:
1. will be loaded with the slide glass of handling the back semen sample and insert under 4 ℃ of envrionment temperatures refrigeration 4-7 minute;
2. taking out the slide glass after the refrigeration, is under 22 ℃ slide glass to be immersed in the hydrochloric acid of 0.08N 7 minutes in room temperature, makes the sperm DNA sex change;
3. the slide glass that will 2. handle through step immersed in the sperm lysate of 20-30 milliliter 20 minutes, the component and the preparation method of sperm lysate are: after getting 6.404 gram Tri(Hydroxymethyl) Amino Methane Hydrochlorides, 1.46 gram ethylenediamine tetraacetic acid (EDTA)s and 11.680 gram sodium-chlor mixing by weight, adding 90 ml distilled waters dissolves, the dithiothreitol dithio that adds 1 milliliter of triton x-100 and 6.17 grams after the dissolving again, be settled to 100 milliliters then, transfer pH value to 7.5;
4. will insert in 20-30 milliliter Tutofusin tris-boric acid-disodium EDTA damping fluid 3 minutes through the slide glass that 3. step was handled;
5. slide glass is used 70%, 90% and 100% dewatering of ethanol successively after taking out in Tutofusin tris-boric acid-disodium EDTA damping fluid, dewatered 2 minutes at every turn;
6. prepare terminal enzyme (DNA) mediated biotin labeling reaction mixture: 1 microlitre deoxyribonucleotide terminal enzyme (DNA), the biotin labeled deoxyuridine triphosphate of 1 microlitre and 98 microlitre level pad mixings are standby;
7. the slide glass after will 5. handling through step, insert 20 microlitres, pH value and be in 7.5 the phosphoric acid buffer and wash 3 times, each 3 minutes, get 100 milliliters of terminal enzyme (DNA) mediated biotin labeling reaction mixtures then and place on the slide glass, add cover glass or seal film and inserted wet box internal reaction dark under 37 ℃ of environment 1 hour;
8. with slide glass by taking out in the wet box, insert and wash 3 times in the phosphoric acid buffer, each 3 minutes, wash the back and drip the Streptavidin that 100 microlitres connect horseradish peroxidase, reaction is 30 minutes under the room temperature;
9. with step 8. in reacted slide glass splash into after developing a film 100 microlitres 3,3 '-diaminobenzidine four hydrochloride working fluids, lucifuge reaction 10 minutes is dried with behind the distilled water flushing 2 times then;
10. at microscopically staining of sperm liquid A liquid is dripped on slide glass for 5 milliliters, sop up unreacted solution after 15 seconds, drip 5 milliliters of staining of sperm liquid B liquid on slide glass again, sop up unreacted solution after 10 seconds, with distillation washing 2 times, dry the back observations again.
The staining of sperm liquid A liquid of step described in 10. is: get 1 water-soluble Yihong of gram and 0.01 gram sodiumazide by weight, insert in 900 ml distilled waters and dissolve, be settled to 1000 milliliters then, be staining of sperm liquid A liquid.
The staining of sperm liquid B liquid of step described in 10. is: get the methylene blue of 0.625 gram and the reddish black A of 0.625 gram by weight, insert 1000 milliliters of pH values and be in 8 the veronal buffer and dissolve, be staining of sperm liquid B liquid after the dissolving, veronal buffer wherein, by weight promptly restraining Veronal sodiums by 1.84 gram veronals and 10.3, insert in 900 ml distilled waters and dissolve, transfer PH to 8.0 to produce after being settled to 1000 milliliters then.
Tutofusin tris-boric acid-disodium EDTA the damping fluid of step described in 5. produced by following raw material and method: get 10.8 gram Tri(Hydroxymethyl) Amino Methane Hydrochlorides, 5.5 gram boric acid, 0.74 gram disodium ethylene diamine tetraacetate by weight, be dissolved in 900 ml distilled waters, be settled to 1000 milliliters, transferring pH value is 7.5.
Step 7. described in phosphoric acid buffer produce by following raw material and method: get 8 gram sodium-chlor, 0.2 gram Repone K, 0.2 gram potassium primary phosphate and 1.15 gram Sodium phosphate dibasics by weight and be dissolved in 900 ml distilled waters, be settled to 1000 milliliters, transferring pH value is 7.4.
The invention has the advantages that: do not need to use expensive instruments such as flow cytometer or fluorescent microscope, only need to adopt the damage of observation by light microscope sperm DNA, thereby make detection method of the present invention for the clinical cheap basis that provides is provided, and, resolution height when the inventive method is observed the sperm DNA damage under opticmicroscope, intuitive is good, halo is clear, judge the accuracy height of sperm quality, simultaneously, because method of the present invention can reduce the generation of false positive results, further improved the reliability of test-results, for the treatment of infertile patients provides reliable clinical diagnosis foundation.Method of the present invention also has advantages of simple operation.
Description of drawings
Fig. 1 and Fig. 2 adopt the observed DNA fragment with spermatozoon of the inventive method, and among the figure, a is a male dna damage sperm, and dark-brown does not have halo; B is negative dna damage sperm, takes on a red color, and halo is clearly arranged, and c is no halo sperm among Fig. 1, is can clearly observe The above results with 200 times of opticmicroscopes.
Embodiment
A kind of method of the present invention with terminal transferase mediated biotin labeling dispersed chromatin detection DNA fragment with spermatozoon, its step is as follows:
1. will be loaded with the slide glass of handling the back semen sample and insert under 4 ℃ of envrionment temperatures refrigeration 4-7 minute;
2. taking out the slide glass after the refrigeration, is under 22 ℃ slide glass to be immersed in the hydrochloric acid of 0.08N 7 minutes in room temperature, makes the sperm DNA sex change;
3. the slide glass that will 2. handle through step immersed in the sperm lysate of 20-30 milliliter 20 minutes, the component and the preparation method of sperm lysate are: after getting 6.404 gram Tri(Hydroxymethyl) Amino Methane Hydrochlorides, 1.46 gram ethylenediamine tetraacetic acid (EDTA)s and 11.680 gram sodium-chlor mixing by weight, adding 90 ml distilled waters dissolves, the dithiothreitol dithio that adds 1 milliliter of triton x-100 and 6.17 grams after the dissolving again, be settled to 100 milliliters then, transfer pH value to 7.5;
4. will insert in 20-30 milliliter Tutofusin tris-boric acid-disodium EDTA damping fluid 3 minutes through the slide glass that 3. step was handled;
5. slide glass is used 70%, 90% and 100% dewatering of ethanol successively after taking out in Tutofusin tris-boric acid-disodium EDTA damping fluid, dewatered 2 minutes at every turn;
6. prepare terminal enzyme (DNA) mediated biotin labeling reaction mixture: 1 microlitre deoxyribonucleotide terminal enzyme (DNA), the biotin labeled deoxyuridine triphosphate of 1 microlitre and 98 microlitre level pad mixings are standby;
7. the slide glass after will 5. handling through step, insert 20 microlitres, pH value and be in 7.5 the phosphoric acid buffer and wash 3 times, each 3 minutes, get 100 milliliters of terminal enzyme (DNA) mediated biotin labeling reaction mixtures then and place on the slide glass, add cover glass or seal film and inserted wet box internal reaction dark under 37 ℃ of environment 1 hour;
8. with slide glass by taking out in the wet box, insert and wash 3 times in the phosphoric acid buffer, each 3 minutes, wash the back and drip the Streptavidin that 100 microlitres connect horseradish peroxidase, reaction is 30 minutes under the room temperature;
9. with step 8. in reacted slide glass splash into after developing a film 100 microlitres 3,3 '-diaminobenzidine four hydrochloride working fluids, lucifuge reaction 10 minutes is dried with behind the distilled water flushing 2 times then;
10. at microscopically staining of sperm liquid A liquid is dripped on slide glass for 5 milliliters, sop up unreacted solution after 15 seconds, drip 5 milliliters of staining of sperm liquid B liquid on slide glass again, sop up unreacted solution after 10 seconds, with distillation washing 2 times, dry the back observations again.
The staining of sperm liquid A liquid of step described in 10. is: get 1 water-soluble Yihong of gram and 0.01 gram sodiumazide by weight, insert in 900 ml distilled waters and dissolve, be settled to 1000 milliliters then and be staining of sperm liquid A liquid.
The staining of sperm liquid B liquid of step described in 10. is: get the methylene blue of 0.625 gram and the reddish black A of 0.625 gram by weight, insert 1000 milliliters of pH values and be in 8 the veronal buffer and dissolve, be staining of sperm liquid B liquid after the dissolving, veronal buffer wherein, by weight promptly restraining Veronal sodiums by 1.84 gram veronals and 10.3, insert in 900 ml distilled waters and dissolve, transfer PH to 8.0 to produce after being settled to 1000 milliliters then.
Tutofusin tris-boric acid-disodium EDTA the damping fluid of step described in 5. produced by following raw material and method: get 10.8 gram Tri(Hydroxymethyl) Amino Methane Hydrochlorides, 5.5 gram boric acid, 0.74 gram disodium ethylene diamine tetraacetate by weight, be dissolved in 900 ml distilled waters, be settled to 1000 milliliters, transferring pH value is 7.5.
Step 7. described in phosphoric acid buffer produce by following raw material and method: get 8 gram sodium-chlor, 0.2 gram Repone K, 0.2 gram potassium primary phosphate and 1.15 gram Sodium phosphate dibasics by weight and be dissolved in 900 ml distilled waters, be settled to 1000 milliliters, transferring pH value is 7.4.
The present invention can observe the sperm of biotin labeled dna damage clearly under opticmicroscope, it is dark-brown and does not have halo, also can be observed clear show diffuse halation do not have a dna damage sperm, its halo that takes on a red color.
5. 1. step be staining of sperm matter diffusion experiment extremely in the inventive method, it can make the depolymerization of sperm nuclear membrane, make staining of sperm matter injury region produce the single stranded DNA segment, and after can removing nucleoprotein, make the sperm nucleus chromatin Structure of dense packing loose, dna circle invests residual nuclear structure, has the sperm of DNA fragment not have halo to form, and does not have the sperm of DNA fragment to have halo to form.
Semen sample of the present invention, its treatment process is: seminal fluid and low melting point agar gel are pressed 1: 2.5 volume ratio mixing, be placed under 36 ℃～38 ℃ and hatched 4～6 minutes, getting 20 microlitres, to drop in through over-richness be on the pretreated slide glass of 0.6% standard agar, covered.
The inventive method step is dewatered to slide glass with ethanol in 5., is slide glass to be inserted in the staining jar dewater, air-dry after, step below continuing, dewatering operation course requires slide glass is in horizontality.
Step of the present invention 3 described in 9., 3 '-diaminobenzidine, four hydrochloride working fluids are with 3, concentrated solution 5 microlitres, 30% hydrogen peroxide, 1 microlitre and phosphoric acid buffer 94 microlitres are mixed and made into 100 microlitres 3 in 3 '-diaminobenzidine, the four hydrochloride colouring reagents boxes, 3 '-diaminobenzidine, four hydrochloride working fluids.
Wet box described in the inventive method is to place waterlogged gauze in box.
Claims (5)
1, a kind of method with terminal transferase mediated biotin labeling dispersed chromatin detection DNA fragment with spermatozoon, it is characterized in that: step is as follows:
1. will be loaded with the slide glass of handling the back semen sample and insert under 4 ℃ of envrionment temperatures refrigeration 4-7 minute;
2. taking out the slide glass after the refrigeration, is under 22 ℃ slide glass to be immersed in the hydrochloric acid of 0.08N 7 minutes in room temperature, makes the sperm DNA sex change;
3. the slide glass that will 2. handle through step immersed in the sperm lysate of 20-30 milliliter 20 minutes, the component and the preparation method of sperm lysate are: after getting 6.404 gram Tri(Hydroxymethyl) Amino Methane Hydrochlorides, 1.46 gram ethylenediamine tetraacetic acid (EDTA)s and 11.680 gram sodium-chlor mixing by weight, adding 90 ml distilled waters dissolves, the dithiothreitol dithio that adds 1 milliliter of triton x-100 and 6.17 grams after the dissolving again, be settled to 100 milliliters then, transfer pH value to 7.5;
4. will insert in 20-30 milliliter Tutofusin tris-boric acid-disodium EDTA damping fluid 3 minutes through the slide glass that 3. step was handled;
5. slide glass is used 70%, 90% and 100% dewatering of ethanol successively after taking out in Tutofusin tris-boric acid-disodium EDTA damping fluid, dewatered 2 minutes at every turn;
6. prepare terminal enzyme (DNA) mediated biotin labeling reaction mixture: 1 microlitre deoxyribonucleotide terminal enzyme (DNA), the biotin labeled deoxyuridine triphosphate of 1 microlitre and 98 microlitre level pad mixings are standby;
7. the slide glass after will 5. handling through step, insert 20 microlitres, pH value and be in 7.5 the phosphoric acid buffer and wash 3 times, each 3 minutes, get 100 milliliters of terminal enzyme (DNA) mediated biotin labeling reaction mixtures then and place on the slide glass, add cover glass or seal film and inserted wet box internal reaction dark under 37 ℃ of environment 1 hour;
8. with slide glass by taking out in the wet box, insert and wash 3 times in the phosphoric acid buffer, each 3 minutes, wash the back and drip the Streptavidin that 100 microlitres connect horseradish peroxidase, reaction is 30 minutes under the room temperature;
9. with step 8. in reacted slide glass splash into after developing a film 100 microlitres 3,3 '-diaminobenzidine four hydrochloride working fluids, lucifuge reaction 10 minutes is dried with behind the distilled water flushing 2 times then;
10. at microscopically staining of sperm liquid A liquid is dripped on slide glass for 5 milliliters, sop up unreacted solution after 15 seconds, drip 5 milliliters of staining of sperm liquid B liquid on slide glass again, sop up unreacted solution after 10 seconds, with distillation washing 2 times, dry the back observations again.
2, a kind of method that detects DNA fragment with spermatozoon with terminal transferase mediated biotin labeling dispersed chromatin according to claim 1, it is characterized in that: the staining of sperm liquid A liquid of step described in 10. is: get 1 water-soluble Yihong of gram and 0.01 gram sodiumazide by weight, insert in 900 ml distilled waters and dissolve, be settled to 1000 milliliters then, be staining of sperm liquid A liquid.
3, a kind of method that detects DNA fragment with spermatozoon with terminal transferase mediated biotin labeling dispersed chromatin according to claim 1, it is characterized in that: the staining of sperm liquid B liquid of step described in 10. is: get the methylene blue of 0.625 gram and the reddish black A of 0.625 gram by weight, insert 1000 milliliters of pH values and be in 8 the veronal buffer and dissolve, be staining of sperm liquid B liquid after the dissolving, veronal buffer wherein, by weight promptly restraining Veronal sodiums by 1.84 gram veronals and 10.3, insert in 900 ml distilled waters and dissolve, transfer PH to 8.0 to produce after being settled to 1000 milliliters then.
4, a kind of method that detects DNA fragment with spermatozoon with terminal transferase mediated biotin labeling dispersed chromatin according to claim 1, it is characterized in that: the Tutofusin tris-boric acid-disodium EDTA damping fluid of step described in 5. produced by following raw material and method: get 10.8 gram Tri(Hydroxymethyl) Amino Methane Hydrochlorides, 5.5 gram boric acid, 0.74 gram disodium ethylene diamine tetraacetate by weight, be dissolved in 900 ml distilled waters, be settled to 1000 milliliters, transferring pH value is 7.5.
5, a kind of method that detects DNA fragment with spermatozoon with terminal transferase mediated biotin labeling dispersed chromatin according to claim 1, it is characterized in that: step 7. described in phosphoric acid buffer produce by following raw material and method: get by weight 8 the gram sodium-chlor, 0.2 the gram Repone K, 0.2 the gram potassium primary phosphate and 1.15 the gram Sodium phosphate dibasics be dissolved in 900 ml distilled waters, be settled to 1000 milliliters, transferring pH value is 7.4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100149335A CN101550447B (en) | 2009-04-30 | 2009-04-30 | Method for detecting sperm DNA fragments by using terminal transferase mediated biotin labeling dispersed chromatin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100149335A CN101550447B (en) | 2009-04-30 | 2009-04-30 | Method for detecting sperm DNA fragments by using terminal transferase mediated biotin labeling dispersed chromatin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101550447A true CN101550447A (en) | 2009-10-07 |
| CN101550447B CN101550447B (en) | 2011-04-20 |
Family
ID=41154936
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009100149335A Expired - Fee Related CN101550447B (en) | 2009-04-30 | 2009-04-30 | Method for detecting sperm DNA fragments by using terminal transferase mediated biotin labeling dispersed chromatin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101550447B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10274485B2 (en) | 2011-07-28 | 2019-04-30 | NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen | Method for detecting biomolecules |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090053722A1 (en) * | 2007-08-21 | 2009-02-26 | David Brown | Sdad assay and uses thereof |
| CN101319251B (en) * | 2008-05-28 | 2010-09-01 | 山东省计划生育科学技术研究所 | Method for detecting spermatozoon DNA fragment with spermatozoon chromatin diffusion experiment |
-
2009
- 2009-04-30 CN CN2009100149335A patent/CN101550447B/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10274485B2 (en) | 2011-07-28 | 2019-04-30 | NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen | Method for detecting biomolecules |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101550447B (en) | 2011-04-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103789202A (en) | Container for collecting nucleic acid preserved at normal temperature | |
| CN101319251B (en) | Method for detecting spermatozoon DNA fragment with spermatozoon chromatin diffusion experiment | |
| CN106980022B (en) | Homogeneous immunoassay method based on target protein induced DNase circulation generation | |
| CN107619775B (en) | Portable nucleic acid detection platform suitable for PCR chromatography | |
| CN215757272U (en) | Nucleic acid amplifier | |
| CN101550447B (en) | Method for detecting sperm DNA fragments by using terminal transferase mediated biotin labeling dispersed chromatin | |
| Gupta et al. | Inter‐and intra‐laboratory standardization of TUNEL assay for assessment of sperm DNA fragmentation | |
| Mallepaddi et al. | Development of loop-mediated isothermal amplification–based lateral flow device method for the detection of malaria | |
| Ajenifuja et al. | Comparison between self sampling and provider collected samples for Human Papillomavirus (HPV) Deoxyribonucleic acid (DNA) testing in a Nigerian facility | |
| Ghasemian et al. | Staphylococcus saprophyticus and Escherichia coli: Tracking from sperm fertility potential to assisted reproductive outcomes | |
| CN106319028A (en) | Kit for detecting mycoplasma urealytium and human mycoplasma | |
| Hayden et al. | Rapid, affordable, and scalable SARS-CoV-2 detection from saliva | |
| RU2362167C1 (en) | Way of diagnostics of male sterility | |
| André et al. | A modified method for COD determination of solid waste, using a commercial COD kit and an adapted disposable weighing support | |
| CN215574673U (en) | Insert type pesticide residue rapid detection kit | |
| CN101907620B (en) | Method for detecting sperm deoxyribonucleic acid fragments and film integrality | |
| CN102839208B (en) | Absolute quantification method for fluorescence in-situ hybrid cell counting | |
| JP2001255323A (en) | Blood separation instrument, blood separation method, method for preparing living body specimen, method for determining living body specimen and living body specimen preserving container | |
| CN102925584B (en) | LAMP (loop-mediated isothermal amplification) detecting kit for turkey trichomonas | |
| CN107201307A (en) | A kind of heparin tube and its application | |
| CN105044356A (en) | Novel prostate-specific antigen detection kit | |
| CN103602725B (en) | Loop-mediated isothermal amplification primer for sesame component, kit and applications of the primer | |
| CN1405562A (en) | Sialidase detection reagent | |
| Li et al. | Construction a novel detection method for Trichomonas vaginalis based on recombinant enzyme polymerase amplification targeting the Actin gene | |
| CN103335934A (en) | Flow cytometry-based sperm motility rate detection reagent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110420 Termination date: 20130430 |