CN101575596B - Method for extracting mitochondria DNA of bull sperm - Google Patents
Method for extracting mitochondria DNA of bull sperm Download PDFInfo
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- CN101575596B CN101575596B CN2009100519489A CN200910051948A CN101575596B CN 101575596 B CN101575596 B CN 101575596B CN 2009100519489 A CN2009100519489 A CN 2009100519489A CN 200910051948 A CN200910051948 A CN 200910051948A CN 101575596 B CN101575596 B CN 101575596B
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Abstract
The invention discloses a method for extracting the mitochondria DNA of bull sperm, which adopts hypotonic solution to lyse the bull sperm. The method does not need expensive machinery equipment, usesconventional reagents, has easy operation, and solves the difficult problem of extracting the mitochondria DNA from a refrigerant bull sperm tubule.
Description
Technical field
The invention belongs to field of genetic engineering, be specifically related to a kind of method of extracting mitochondria DNA of bull sperm.
Background technology
Mitochondrial DNA (mtDNA) is to be independent of the outer genetic material of nuclear gene group in the phalangeal cell, is the deoxyribonucleotide that a double-stranded circular, size are about 16KB and energy self-replacation.Plastosome is at apoptosis, performance plays an important role in aging and the programmed cell death, in livestock industry, milk yield of milk cow and fat yield, meat of beef cattle and calving rate etc. may all relevant with mtDNA (Tamassia M, et al.Evidence of oocyte donorcow effect over oocyte production and embryo development in vitro.Reproduction, 2003,126 (5): 629～637), also find relevant (the Tamassia M of the external Embryo Production of mtDNA haplotype and ox (IVP) efficient recently, Nuttinck F, Reynier P, et al. In vitro embyro production efficiency incattle and its association with oocyte adenosine triphosphat
Common from whole blood or tissue the method method (Feng Kai, Liu Xing, the Jiang Jianxin that introduce such as Feng Kai for example of extracting DNA.Introduce a kind of from whole blood the method for extracting DNA. Third Military Medical University's journal, 2004,26 (4): 368～369), comprise the PBS washing, Proteinase K and stain remover digestion, phenol and chloroform purifying, ethanol sedimentation, TE dissolving.This method is because the stain remover that uses, be that SDS can not digest the cytolemma of sperm and the cracking sperm, and is not suitable for extracting mitochondria of sperms DNA.
The plastosome of sperm is positioned at the afterbody stage casing, for the swing of afterbody flagellum provides energy.Contain complex materials such as lecithality glycerine in the refrigerated Niu Jingzi tubule, organize Mitochondrial DNA method and infeasible with common extracting, and the plastosome of sperm merges under the help of microfilament, and it and flagellum are formed mitochondrial sheath jointly, has further increased the difficulty of extracting mitochondria of sperms DNA.An ovocyte contains up to 10
5To 10
8Plastosome NDA copy, and only contain 100 Mitochondrial DNAs copies in a The mature sperm, so extracting DNA was difficult in ten minutes from sperm.
Therefore the method that does not have effective extracting mitochondria of sperms DNA at present will seek out a large amount of DNA, at first separate mitochondria.At present approaching therewith is extracting Mitochondrial DNA from tissue, it mainly is by differential centrifugation separate mitochondria (Shang Tao etc., the mitochondrial method research of extraction separation lungs, Hebei medicine, 2006,28 (4): 252-253), centrifugal step by step by low speed in the medium of density homogeneous to high speed, be used to separate the cell and the organoids of different sizes, the settled order of organoid is followed successively by in differential centrifugation: nuclear, plastosome, lysosome and peroxysome, endoplasmic reticulum and high matrix, be ribosome at last.Differential centrifugation only is used to separate greatly different in size cell, the isolated cell devices that are used for more.The organoid initial gross separation often can be needed further by the centrifugal capable again separation and purification of density level bands by differential centrifugation.And this centrifugation needs ultracentrifuge, and the machinery equipment cost is huge, and needs extra reagent, is not suitable for applying.
Hypotonic technology common application is observed in microcytoscope, does not see that at present report is arranged but be used for the extracting Mitochondrial DNA.
Summary of the invention
The present inventor finds under study for action, behind the employing hypotonic solution cracking Niu Jingzi, re-uses the method for conventional extracting Mitochondrial DNA, can successfully obtain mitochondria DNA of bull sperm.
Therefore, purpose of the present invention just is to provide a kind of method of extracting mitochondria DNA of bull sperm.
The method of extracting mitochondria DNA of bull sperm of the present invention comprises and adopts hypotonic solution cracking Niu Jingzi.
According to a specific embodiment of the present invention, described hypotonic solution preferably osmotic pressure at the solution of 0～100Osm.
According to the present invention, the described hypotonic solution cracked time is 0.5～24 hour.
According to the present invention, described hypotonic solution cracked temperature is 10～50 ℃.
According to the present invention, the Niu Jingzi after the hypotonic solution cracking further adopts the step of conventional extracting Mitochondrial DNA can obtain mitochondria DNA of bull sperm, and these steps comprise:
1, the centrifugal supernatant of abandoning;
2, adding STE suspends;
3, the SDS digesting protein that adds Proteinase K and 10%;
4, add saturated phenol and chloroform/primary isoamyl alcohol removing protein, get supernatant after centrifugal;
5, supernatant adds the NaAc mixing, adds the dehydrated alcohol precipitation again;
6, the centrifugal supernatant of abandoning precipitates with washing with alcohol again;
7, centrifugal back room temperature is air-dry.
Method of the present invention need not expensive machinery equipment, use conventional reagent, working method is simple, contain complex materials such as lecithality glycerine in the refrigerated Niu Jingzi tubule, organize Mitochondrial DNA method and infeasible with common extracting, and the plastosome of sperm merges under the help of microfilament, it and flagellum are formed mitochondrial sheath jointly, further increased the difficulty of extracting mitochondria of sperms DNA, the invention solves the difficult problem of extracting Mitochondrial DNA from refrigerated Niu Jingzi tubule, have important biological significance and actual using value.
Description of drawings
Fig. 1 is that osmotic pressure is 0Osm solution cracking Niu Jingzi (20 ℃ of room temperatures, 1h) electrophoresis result of back extracting mitochondria DNA of bull sperm.
Fig. 2 is that osmotic pressure is 25Osm solution cracking Niu Jingzi (20 ℃ of room temperatures, 1h) electrophoresis result of back extracting mitochondria DNA of bull sperm.
Fig. 3 is that osmotic pressure is 50Osm solution cracking Niu Jingzi (20 ℃ of room temperatures, 1h) electrophoresis result of back extracting mitochondria DNA of bull sperm.
Fig. 4 is that osmotic pressure is 75Osm solution cracking Niu Jingzi (20 ℃ of room temperatures, 1h) electrophoresis result of back extracting mitochondria DNA of bull sperm.
Fig. 5 is that osmotic pressure is 100Osm solution cracking Niu Jingzi (20 ℃ of room temperatures, 1h) electrophoresis result of back extracting mitochondria DNA of bull sperm.
Fig. 6 is that osmotic pressure is 125Osm solution cracking Niu Jingzi (20 ℃ of room temperatures, 1h) electrophoresis result of back extracting mitochondria DNA of bull sperm.
Fig. 7 is that osmotic pressure is 150Osm solution cracking Niu Jingzi (20 ℃ of room temperatures, 1h) electrophoresis result of back extracting mitochondria DNA of bull sperm.
Fig. 8 is that osmotic pressure is the electrophoresis result of the extracting mitochondria DNA of bull sperm of (1h) under the different cracking temperatures of 50Osm solution.
Fig. 9 is that osmotic pressure is the electrophoresis result of 50Osm solution extracting mitochondria DNA of bull sperm of (20 ℃ of room temperatures) under the different cracking times.
Figure 10 is that the NlaIII enzyme of Niu Jingzi mtDNA is cut rear electrophoresis analytical results (pH 8.0 for 1% agarose gel electrophoresis, TAE damping fluid, voltage 150V, 40 minutes).
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples only are used to the present invention is described but not are used to limit scope of the present invention.
In following examples, the Niu Jingzi of use is available from Shanghai Bright Dairy ﹠ Food Co., Ltd..
STE prescription: 50Mm Tris, pH7.5,1mM EDTA, pH8.0,0.1M NaCl.
Proteinase K: available from CALBIOCHEM company.
In following examples, as non-special instruction, agents useful for same is all available from TAKARA company.
For groping the osmotic pressure of cracking sperm the best, (normal physiological brinish osmotic pressure is 320Osm with solution (320Osm), being lower than 320Osm and being called hypotonic solution) dilution is for different osmotic pressure, examine under a microscope respectively, if sperm breaks, tail stretches, and is labeled as "+", if sperm does not break and is labeled as "-", observations is as shown in table 1.
Determining of table 1, suitable osmotic pressure
As shown in Table 1, ox sperm performance state difference under different osmotic, Niu Jingzi can break at 0～100Osm, does not then break at＞100Osm, and the osmotic pressure of determining to be suitable for cracking Niu Jingzi thus is 0～100Osm.
At first get the tubule of storing frozen seminal fluid, with the warm water recovery, then according to the following steps extracting mitochondria DNA of bull sperm:
1) Fu Su seminal fluid is abandoned supernatant after 2000rpm is centrifugal 10 minutes;
2) add the hypotonic solution that 1ml osmotic pressure is followed successively by 0Osm, 25Osm, 50Osm, 75Osm, 100Osm, 125Osm, 150Osm respectively, mixing for several times turns upside down, leave standstill 1 hour with the cracking sperm in 20 ℃ of room temperatures, 8000rpm abandons supernatant after centrifugal 5 minutes then;
3) precipitation adds 320 μ l STE suspension, blows and beats mixing with pipettor;
4) SDS of adding 5 μ l Proteinase Ks (20mg/ml) and 10 μ l 10% in the suspension puts into 37 ℃ of water-baths and spends the night behind abundant mixing on the vibrator;
5) add saturated phenol of 200 μ l and the abundant mixing of 200 μ l chloroform/primary isoamyl alcohol in the reaction solution, centrifugal 10 minutes of 13000rpm gets supernatant;
6) after supernatant liquor adds 40 μ l NaAc mixings earlier, add the dehydrated alcohol of 800 μ l precoolings again, left standstill 2 hours in-20 ℃ of refrigerators, 13000rpm is centrifugal 10 minutes then, adds 800 μ l, 80% washing with alcohol more once;
7) centrifugal back room temperature air-dry (about 1 hour) adds 20 μ l distilled waters and dissolves standby.
3.1, design of primers
As follows at the primer that the design of ox Mitochondrial DNA is special:
P1：5`-CTGCAGTCTCACCATCAACC-3`
P2：5`-GTGTAGATGCTTGCATGTGTAAGT-3`
Purpose sheet segment length 1094bp.
3.2, pcr amplification
With embodiment 2 extractive mitochondria DNA of bull sperm is that template is carried out pcr amplification:
Reaction system (25 μ l): template 1 μ l, primer (10 μ M) 1 μ l, ExTaq enzyme 0.2 μ l, Buffer 2.5 μ l, dNTP (2.5mM) 2 μ l, ddH2O 17.3 μ l.
Reaction conditions: 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 1min; 58 ℃ of renaturation 1min, 72 ℃ are extended 2min, circulate 32 times; 72 ℃, extend 10min again.
Gained PCR product is carried out agarose gel electrophoresis, and the result is shown in Fig. 1～7.
By the result of Fig. 1～7 as can be known, be the cracking of 0～100Osm solution through osmotic pressure after, extracting easily is to mitochondria DNA of bull sperm, and 125Osm and 150Osm solution can not cracking Niu Jingzi, therefore can not get mitochondria DNA of bull sperm.
3.3, restriction enzyme cuts evaluation
By restriction enzyme NlaIII the PCR product is carried out enzyme and cut, the base sequence CATG of NlaIII in can the specific recognition mitochondria DNA of bull sperm, and open phosphodiester bond at the two ends of sequence and produce sticky end.
Enzyme tangent condition: buffer 2 μ l, NlaIII 0.2 μ l, PCR product 5 μ l, distilled water 12.8 μ l, reaction system is totally 20 μ l, 37 ° of water-baths 8 hours.
The gained enzyme is cut product carry out electrophoresis, 1% sepharose, the TAE damping fluid, pH 8.0, voltage 150V, 40 minutes, the result was as shown in figure 10.
By the result of Figure 10 as can be known, enzyme is cut each segmental size of back and is added up and conform to further proof amplification mitochondria DNA of bull sperm success with the segmental size of step 3.2 amplification PCR.
In order further to grope temperature and time following experiment is carried out in the influence of hypotonic solution cracking Niu Jingzi extracting Mitochondrial DNA:
4.1, temperature
Recovery sperm at first, use osmotic pressure to be 50Osm solution (80 ℃, 65 ℃, 50 ℃, 35 ℃, 10 ℃, 4 ℃) cracking Niu Jingzi 1 hour under differing temps then, according to embodiment 2 described step extracting mitochondria DNA of bull sperm, carry out the detection of mitochondria DNA of bull sperm according to embodiment 3 described methods, the result as shown in Figure 8.
By the result of Fig. 8 as can be known, can extracting obtain Mitochondrial DNA at 10～50 ℃ of cracked Niu Jingzi.
4.2, the time
Recovery sperm at first, use then osmotic pressure as 50Osm solution at 20 ℃ of following cracking Niu Jingzi of room temperature (5 minutes, 30 minutes, 24 hours, 48 hours), according to embodiment 2 described step extracting mitochondria DNA of bull sperm, carry out the detection of mitochondria DNA of bull sperm according to embodiment 3 described methods, the result as shown in Figure 9.
By the result of Fig. 9 as can be known, 30 minutes～24 hours Niu Jingzi of cracking at room temperature can extracting obtain Mitochondrial DNA.
By the result of Fig. 8 and Fig. 9 as can be seen, the top condition of hypotonic solution cracking Niu Jingzi is 10～50 ℃ and left standstill 30 minutes～24 hours.
Claims (1)
1. the method for an extracting mitochondria DNA of bull sperm, it is characterized in that: described method comprises employing hypotonic solution cracking Niu Jingzi, the described hypotonic solution cracked time is 0.5～24 hour, described hypotonic solution cracked temperature is 10～50 ℃, describedly obtains mitochondria DNA of bull sperm through hypotonic solution cracked Niu Jingzi by following steps:
1) the centrifugal supernatant of abandoning;
2) adding STE suspends;
3) add the SDS digesting protein of Proteinase K and 10%;
4) add saturated phenol and chloroform/primary isoamyl alcohol removing protein, get supernatant after centrifugal;
5) supernatant adds the NaAc mixing, adds the dehydrated alcohol precipitation again;
6) the centrifugal supernatant of abandoning precipitates with washing with alcohol again;
7) centrifugal back room temperature is air-dry.
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| CN102199594B (en) * | 2009-08-05 | 2012-09-05 | 公安部物证鉴定中心 | Method for extracting and purifying semen DNA |
| CN106244585B (en) * | 2016-10-11 | 2019-11-22 | 皖南医学院 | A kind of extracting method of simple and efficient oncomelania mitochondrial genomes DNA |
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| CN101134772A (en) * | 2007-07-25 | 2008-03-05 | 中国农业大学 | Method for separating membrane protein of sucking sperm rear head and uses thereof |
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| CN101134772A (en) * | 2007-07-25 | 2008-03-05 | 中国农业大学 | Method for separating membrane protein of sucking sperm rear head and uses thereof |
Non-Patent Citations (5)
| Title |
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| Patrick K.Schoff, et al.Adenylate kinase activity in ejaculated bovine sperm flagella.《The journal of biological chemistry》.1989,第264卷(第11期),6086-6091. * |
| 夏玉玲.动物线粒体DNA提取的原理和方法.《蚕学通讯》.2002,第22卷(第3期),24-29. * |
| 李伟文等.线粒体DNA提取方法的比较.《国外医学（分子生物学分册）》.2003,第25卷(第3期),191-193. * |
| 郑英等.线粒体DNA与人精子活力间的相关性分析.《解剖学报》.2000,第31卷(第1期),34-38. * |
| 闫华超.动物线粒体DNA提取简易流程及其优化.《生物技术通讯》.2007,第18卷(第1期),95-97. * |
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Effective date of registration: 20190218 Address after: Room 1807-6, No. 1, Lane 600, Tianshan Road, Changning District, Shanghai 200051 Patentee after: Shanghai Taohua Biomedical Technology Partnership (Limited Partnership) Address before: 200040 No. 24, 1400 Lane, Beijing West Road, Shanghai Co-patentee before: Shanghai Taotao Transgene Engineering Co., Ltd. Patentee before: Shanghai City Children Hospital |