CN101717772B - Method for extracting and purifying animal mitochondria DNA - Google Patents
Method for extracting and purifying animal mitochondria DNA Download PDFInfo
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- CN101717772B CN101717772B CN2009102312721A CN200910231272A CN101717772B CN 101717772 B CN101717772 B CN 101717772B CN 2009102312721 A CN2009102312721 A CN 2009102312721A CN 200910231272 A CN200910231272 A CN 200910231272A CN 101717772 B CN101717772 B CN 101717772B
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Abstract
The invention discloses a method for extracting animal mitochondria DNA, which has the innovativeness that a DNasel digestion step is added on the basis of alkaline denaturation to eliminate residual nuclear DNA; and an RNase digestion step is added to remove residual RNA. The mitochondria DNA extracted by the method has high purity. The electrophoretic detection shows that an electrophoretic band is a clear, regular and uniform band; and the background is clear, and the conditions such as the trailing of macromolecular DNA and front-end RNA and the like do not occur near a sample application pore.
Description
Technical field
The invention belongs to molecular biology nucleic acid extraction purification technique field, specifically relate to a kind of extraction and purification process of animal mitochondria DNA.
Background technology
Existing animal mitochondria DNA extractive technique mainly is an alkaline denaturation.This technology adopts differential centrifugation to extract plastosome; SDS alkaline denaturation cracking plastosome; Phenol extraction process purifying mtDNA.This technological outstanding feature is easy to be quick, therefore should technology be used widely in fields such as biology, medical science and legal medical expert's evaluations.
There are macromole DNA (residual nuclear DNA) and front end that RNA hangover situation such as (residual RNA) is arranged near the point sample hole owing to be everlasting when using the Mitochondrial DNA electrophoresis of this technology extraction; The high molecular biology research that can not meet the demands is cut and order-checking etc. like pcr amplification, enzyme.Therefore, only on this technological basis, improve, improve product gas purity, could satisfy the needs of high-level molecular biology research, provide safeguard for the research of China's field of biology is in line with international standards.
Summary of the invention
To the deficiency of prior art, the purpose of this invention is to provide a kind of highly purified animal mitochondria DNA and extract and purification process.
Technical scheme of the present invention is following: a kind of process for extracting of animal mitochondria DNA, its novelty are, on the basis of alkaline denaturation, have increased the DNaseI digestion step, to eliminate the residual of nuclear DNA; Increased the RNase digestion step, to remove the residual of RNA.Concrete steps are following:
(1) gets animal tissues, add 5-50mLSE liquid, after homogenate, the filtration, suck centrifuge tube;
(2) the centrifugal 10-15min of 1500r/min gets supernatant, under 0 ℃ of condition; The centrifugal 20-30min of 12000r/min stays deposition, adds STM liquid (or SE liquid) and suspends; Adding solution D, to make final concentration be 100 μ g/mL, and 37 ℃ of temperature are bathed 30min (to remove nuclear DNA), the centrifugal 10min of 12000r/min; Add SE liquid (or STM liquid) suspension plastosome, go to the 1.5mLEppendorf pipe then;
(3) the centrifugal 8-10min of 12000r/min abandons supernatant, adds 150 μ L solution A mixings, adds the solution B of 300 μ L new systems, mixing, and ice bath 10min adds 225 μ L solution C, mixing, ice bath 25-60min, the temperature of solution C is 4 ℃;
(4) the centrifugal 6min of 12000r/min gets supernatant, adds the equal-volume water-saturated phenol, behind the room temperature vibration 15min, adds and the isopyknic phenol of supernatant, chloroform and primary isoamyl alcohol mixed solution, mixing, the volume ratio of phenol, chloroform and primary isoamyl alcohol 25: 24: 1 again;
(5) the centrifugal 8-10min of 12000r/min, the water intaking phase adds the absolute ethyl alcohol mixing of 2-2.5 times of volume, and the centrifugal 10min of 12000r/min gets deposition, and using temperature is 0 ℃ 70% washing with alcohol, the centrifugal 2min of 12000r/min, seasoning;
(6) the gained animal mitochondria DNA is added Tris-EDTA damping fluid dissolving, adding solution E, to make final concentration be 20ug/mL, and-20 ℃ frozen subsequent use.
Said: SE liquid: 0.25mol/L sucrose, 30mmol/LTris-HCl, 10mmol/L Na
2EDTA, 2.5mmol/L CaCl
2, pH:7.3-8.1; STM liquid: 0.25mol/L sucrose, 10mmol/LTris-HCl, 0.5mmol/L MgCl
2, pH:8.0; Solution A: 10mmol/LTris-HCl, 10mmol/LNa
2EDTA, 0.15mol/L NaCl, pH:8.0; Solution B: 1%SDS contains 0.2N NaOH, and the time spent joins at present; Solution C: KAc solution, 294gKAc, 50mL 90% formic acid adds water to 1000mL, pH:5.5; Solution D: DNaseI solution, with the preparation of TE damping fluid, concentration 5mg/mL; Solution E: RNaseA solution, with TE damping fluid dissolving RNaseA, the concentration preparation 100mL with 1mg/mL boils 10-15min, is divided into 500 μ l/ pipe, is stored in-20 ℃.
Aforesaid extraction and purification process, preferred scheme is that said step (1) is got animal tissues's rinsing in SE liquid, shredded; Add 5-50mLSE liquid, with homogenate about electric homogenizer 1500r/min 10-12 time, each 5 seconds; After the filtration, homogenate is sucked in the centrifuge tube.
Aforesaid extraction and purification process, preferred scheme is that temperature is 0 ℃ during said step (1) homogenate.
Aforesaid extraction and purification process, preferred scheme are that animal tissues is flesh tissue, frozen tissue or the formaldehyde immersion tissue of animal in the said step (1).
Aforesaid extraction and purification process, preferred scheme be, said animal tissues is liver or the gonadal tissue of fish, liver, sexual gland or the muscle tissue of Rana nigromaculata.
Aforesaid extraction and purification process, preferred scheme is the saturated phenol pH8.0 of said step (4).
Aforesaid extraction and purification process; Preferred scheme is, step (5) gained animal mitochondria DNA is added solution E make final concentration 20ug/mL, bathes 60min 37 ℃ of temperature; Repetitive operation step again (4) and (5), product is in-20 ℃ frozen subsequent use (more effectively removing RNA).
Aforesaid extraction and purification process, preferred scheme is that step (5) products obtained therefrom also can place 4 ℃ of refrigerator dryings, carries out subsequent step again.
Compared with prior art, excellent effect of the present invention is:
1, extracts the Mitochondrial DNA purity height that obtains.
2, electrophoresis detection shows: electrophoretic band is clear, neat, a uniform band; Background is clear, and not seeing near the point sample hole has situation such as macromole DNA and front end RNA hangover.
3, the OD value analyze to show: OD260/OD280 value is 1.78-1.82, the requirement that the enzyme that can satisfy Mitochondrial DNA is cut and sequencing analysis etc. is studied.
Description of drawings
Fig. 1. the electrophorogram of embodiment 1 gained Mitochondrial DNA.
Fig. 2. the simple and easy schematic flow sheet of the extraction of animal mitochondria DNA of the present invention and purification process.
Embodiment
Below in conjunction with embodiment and accompanying drawing technical scheme of the present invention is done further to specify.But the present invention does not receive the restriction of these specific embodiments.If amplifying, extensive enforcement, equal proportion get final product.
Raw materials used among the embodiment (or equipment) is the conventional articles for use in this area, and it all can be bought from market.Specify as follows: SE liquid (homogenate buffer): 0.25mol/L sucrose, 30mmol/LTris-HCl, 10mmol/L Na
2EDTA, 2.5mmol/LCaCl
2, pH:7.3-8.1; STM liquid: 0.25mol/L sucrose, 10mmol/LTris-HCl, 0.5mmol/L MgCl
2, pH:8.0; Solution A (TEN): 10mmol/LTris-HCl, 10mmol/LNa
2EDTA, 0.15mol/L NaCl, pH:8.0; Solution B: 1%SDS contains 0.2N NaOH (time spent newly prepares with 10%SDS and 1N NaOH); Solution C: KAc solution, 294gKAc, 50mL 90% formic acid adds water to 1000mL, pH:5.5; Solution D: DNaseI solution, with the preparation of TE damping fluid, concentration 5mg/mL; Solution E: RNaseA solution, with TE damping fluid dissolving RNaseA, the concentration preparation 100mL with 1mg/mL boils 10-15min, is divided into 500 μ l/ pipe, is stored in-20 ℃.
TE damping fluid wherein: promptly (pH8.0), available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, the used KAc of preparation KAc solution is available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd for 10mM Tris, 1mM EDTA for Tris-EDTAbuffer.
Embodiment 1: the extraction of fishing line mitochondrial DNA.
(1) gets the liver organization of 5-6g fish, rinsing in a small amount of SE solution, shred, add about 50mLSE, with electric homogenizer 1500r/min homogenate 12 times up and down.
(2) homogenate is drawn supernatant with the centrifugal 15min of 1000r/min, the centrifugal 20min of 12000r/min, deposition is plastosome.Add 4mLSTM liquid suspension plastosome, adding solution D, to make final concentration be 100 μ g/mL (to remove nuclear DNA), and 37 ℃ of temperature are bathed, 30min, and the centrifugal 10min of 12000r/min abandons supernatant; Add 4mLSE liquid suspension plastosome, change over to again in 4 1.5mL Eppendorf pipes.
(3) the centrifugal 10min of 12000r/min abandons supernatant, and every pipe adds 150 μ l solution A, will precipitate suspend after, the solution B mixing that each adds the new preparation of 300 μ l behind the ice bath 10min, respectively adds 225 μ l cold soln C (4 ℃), mixing, ice bath 30min again.
(4) the centrifugal 6min of 12000r/min draws supernatant, adds isopyknic water-saturated phenol, behind the room temperature vibration 15min, adds the phenol-chloroform-primary isoamyl alcohol (volume ratio 25: 24: 1) of 1 times of volume of former supernatant, fully mixing.
(5) the centrifugal 8min of 12000r/min draws water, adds 2.5 times of volume absolute ethyl alcohol mixing ice bath 30min.The centrifugal 10min of 12000r/min is with 70% cold ethanol (0 ℃) washing precipitation, the centrifugal 2min of 12000r/min, seasoning.
(6) add the dissolving of an amount of volume Tris-EDTA damping fluid after, add solution E (final concentration 20 μ g/mL) ,-20 ℃ frozen subsequent use.
The OD260/OD280 value of present embodiment gained mitochondrial DNA is 1.78, and pcr amplification, the enzyme that can satisfy Mitochondrial DNA cut and the requirement of research such as sequencing analysis.
Fig. 1 is the electrophorogram of Mitochondrial DNA that present embodiment obtains.Through the visible electrophoretic band of Fig. 1 is clear, neat, a uniform band; Background is clear, and not seeing near the point sample hole has situation such as macromole DNA and front end RNA hangover.Explain that not see nuclear DNA and RNA in the Mitochondrial DNA that extracts residual.
Embodiment 2: the Mitochondrial DNA of the frog extracts purifying.
(1) homogenate: liver or the muscle of getting 2～15g Rana nigromaculata shred in a small amount of SE homogenate buffer, add about 20～30mLSE liquid, with homogenate about the electric homogenizer 1500r/min 10 times, each 5 seconds, after the filtration, homogenate are sucked in the centrifuge tube.
(2) purifying plastosome: the centrifugal 15min of 1500r/min gets supernatant for the first time.For the second time the centrifugal 20min of 12000r/min gets deposition, adds 2mLSTM liquid suspension plastosome, and adding solution D, to make final concentration be 100 μ g/mL, and 37 ℃ of temperature are bathed; 30min, the centrifugal 10min of 12000r/min abandons supernatant; Add 4mLSE liquid and suspend, go to 4 1.5mL Eppendorf pipes then, every pipe 1mL.
(3) the centrifugal 8min of 12000r/min abandons supernatant, and every pipe adds 150 μ L solution A will precipitate the solution B that adds 300 μ L new systems after the piping and druming evenly, mixing, and ice bath 10min respectively adds 225 μ L cold soln C, mixing, ice bath 60min.
(4) the centrifugal 6min of 12000r/min gets supernatant, adds isopyknic water-saturated phenol, behind the room temperature vibration 15min, adds fully mixing of the isopyknic phenol-chloroform-primary isoamyl alcohol of former supernatant (volume ratio 25: 24: 1) again.
(5) the centrifugal 8min of 12000r/min, the water intaking phase adds the absolute ethyl alcohol mixing of two volumes, ice bath 30min.The centrifugal 10min of 12000r/min.With 70% cold ethanol (0 ℃) washing precipitation.The centrifugal 2min of 12000r/min.Seasoning 3～4 hours or 4 ℃ of refrigerator overnight finish-dryings.
(6) add the dissolving of an amount of volume Tris-EDTA damping fluid after, add solution E (final concentration 20ug/mL), 37 ℃, temperature is bathed 60min, repetitive operation step (4) and (5), refrigerator is frozen subsequent use.
The OD260/OD280 value of present embodiment gained mitochondrial DNA is 1.80, and the enzyme that can satisfy Mitochondrial DNA is cut and the requirement of research such as sequencing analysis.
Embodiment 3: the honeybee Mitochondrial DNA extracts purifying.
(1) gets fresh honeybee or alcohol-pickled sample and in a small amount of SE homogenate buffer, shred, add about 2～5mLSE liquid,, each 5 seconds, after the filtration, homogenate is sucked in the centrifuge tube with homogenate about the electric homogenizer 1500r/min 10 times.
(2) the centrifugal 15min of 1500r/min gets supernatant for the first time.For the second time the centrifugal 20min of 12000r/min gets deposition, adds 4mLSE liquid suspension plastosome, and adding solution D, to make final concentration be 100 μ g/mL (to remove nuclear DNA); 37 ℃ of temperature are bathed 30min, the centrifugal 10min of 12000r/min; Abandon supernatant; Add 2mLSTM liquid suspension plastosome, go to 2 1.5mLEppendorf pipes then, every pipe 1mL.
(3) the centrifugal 8min of 12000r/min abandons supernatant, and every pipe adds 150 μ L solution A will precipitate the solution B that adds 300 μ L new systems after the piping and druming evenly, mixing, and ice bath 10min respectively adds 225 μ L cold soln C, mixing, ice bath 60min.
(4) the centrifugal 6min of 12000r/min gets supernatant, adds isopyknic water-saturated phenol solution, behind the room temperature vibration 15min, adds and the abundant mixing of the isopyknic phenol-chloroform-primary isoamyl alcohol of former supernatant (volume ratio 25: 24: 1) again.
(5) the centrifugal 8min of 12000r/min, the water intaking phase adds the absolute ethyl alcohol mixing of two volumes, ice bath 30min.The centrifugal 10min of 12000r/min.With 70% cold ethanol (0 ℃) washing precipitation.The centrifugal 2min of 12000r/min.Seasoning 3～4 hours or 4 ℃ of refrigerator overnight finish-dryings.
(6) add the dissolving of an amount of volume Tris-EDTA damping fluid after, add an amount of volume Tris-EDTA damping fluid dissolving after, add solution E (final concentration 20 μ g/mL).Add 25 μ LTE dissolving, refrigerator is frozen subsequent use.
The OD260/OD280 value of present embodiment gained mitochondrial DNA is 1.82, and the enzyme that can satisfy Mitochondrial DNA is cut and the requirement of research such as sequencing analysis.
Embodiment 4: the extraction of fishing line mitochondrial DNA.Different is the gonadal tissue with fish with embodiment 1, and temperature is 0 ℃ during step (1) homogenate.
Embodiment 5: the Mitochondrial DNA of the frog extracts purifying.Different is the gonadal tissue with Rana nigromaculata with embodiment 2, the saturated phenol pH8.0 of step (4).
Claims (8)
1. the extraction of an animal mitochondria DNA and purification process is characterized in that, concrete steps are following:
(1) gets animal tissues, add 5-50mLSE liquid, after homogenate, the filtration, suck centrifuge tube;
(2) the centrifugal 10-15min of 1500r/min gets supernatant, under 0 ℃ of condition; The centrifugal 20-30min of 12000r/min stays deposition, adds STM liquid and suspends; Adding solution D, to make final concentration be 100 μ g/mL, and 37 ℃ of temperature are bathed 30min, the centrifugal 10min of 12000r/min; Add SE liquid and suspend, go to 1.5mL Eppendorf pipe then;
(3) the centrifugal 8-10min of 12000r/min abandons supernatant, adds 150 μ L solution A mixings, adds the solution B of 300 μ L new systems, mixing, and ice bath 10min adds 225 μ L solution C, mixing, ice bath 25-60min, the temperature of solution C is 4 ℃;
(4) the centrifugal 6min of 12000r/min gets supernatant, adds the equal-volume water-saturated phenol, behind the room temperature vibration 15min, adds and the isopyknic phenol of supernatant, chloroform and primary isoamyl alcohol mixed solution mixing, the volume ratio of phenol, chloroform and primary isoamyl alcohol 25: 24: 1 again;
(5) the centrifugal 8-10min of 12000r/min, the water intaking phase adds the absolute ethyl alcohol mixing of 2-2.5 times of volume, and the centrifugal 10min of 12000r/min gets deposition, and using temperature is 0 ℃ 70% washing with alcohol, the centrifugal 2min of 12000r/min, seasoning;
(6) the gained animal mitochondria DNA is added Tris-EDTA damping fluid dissolving, adding solution E, to make final concentration be 20ug/mL, and-20 ℃ frozen subsequent use;
Said: SE liquid: 0.25mol/L sucrose, 30mmol/LTris-HCl, 10mmol/L Na
2EDTA, 2.5mmol/L CaCl
2, pH:7.3-8.1; STM liquid: 0.25mol/L sucrose, 10mmol/LTris-HCl, 0.5mmol/L MgCl
2, pH:8.0; Solution A: 10mmol/LTris-HCl, 10mmol/LNa
2EDTA, 0.15mol/L NaCl, pH:8.0; Solution B: 1%SDS contains 0.2N NaOH, and the time spent joins at present; Solution C: KAc solution, 294gKAc, 50mL 90% formic acid adds water to 1000mL, pH:5.5; Solution D: DNaseI solution, with the preparation of TE damping fluid, concentration 5mg/mL; Solution E: RNaseA solution, with TE damping fluid dissolving RNaseA, the concentration preparation 100mL with 1mg/mL boils 10-15min, is divided into 500 μ l/ pipe, is stored in-20 ℃.
2. described extraction of claim 1 and purification process is characterized in that, said step (1) is got animal tissues's rinsing in SE liquid, shredded; Add 5-50mLSE liquid, with homogenate about electric homogenizer 1500r/min 10-12 time, each 5 seconds; After the filtration, homogenate is sucked in the centrifuge tube.
3. claim 1 or 2 described extraction and purification process is characterized in that, temperature is 0 ℃ during said step (1) homogenate.
4. described extraction of claim 1 and purification process is characterized in that, animal tissues is flesh tissue, frozen tissue or the formaldehyde immersion tissue of animal in the said step (1).
5. described extraction of claim 4 and purification process is characterized in that, said animal tissues is liver or the gonadal tissue of fish, liver, sexual gland or the muscle tissue of Rana nigromaculata.
6. described extraction of claim 1 and purification process is characterized in that, the saturated phenol pH8.0 of said step (4).
7. described extraction of claim 1 and purification process is characterized in that, step (5) gained animal mitochondria DNA is added solution E make final concentration 20ug/mL, bathe 60min 37 ℃ of temperature, repetitive operation step again (4) and (5), and drying prods is ℃ frozen subsequent use again-20.
8. described extraction of claim 1 and purification process is characterized in that, step (5) products obtained therefrom places 4 ℃ of refrigerator dryings.
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| CN107841483B (en) * | 2017-11-13 | 2021-08-03 | 珠海龄值生物科技有限公司 | Method for extracting animal tissue mitochondria |
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