CN101338310A - Process for extracting cytoplasm DNA - Google Patents
Process for extracting cytoplasm DNA Download PDFInfo
- Publication number
- CN101338310A CN101338310A CNA200810070110XA CN200810070110A CN101338310A CN 101338310 A CN101338310 A CN 101338310A CN A200810070110X A CNA200810070110X A CN A200810070110XA CN 200810070110 A CN200810070110 A CN 200810070110A CN 101338310 A CN101338310 A CN 101338310A
- Authority
- CN
- China
- Prior art keywords
- adds
- solution
- centrifugal
- minutes
- dna
- 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for extracting DNA of a cell which adopts the following processes: a single cell or a peripheral white blood cell-the cracking of a cell and a mitochondria-the deformation and renaturation of DNA-the extraction and purifying of the DNA; the cytoplasm DNA is directly extracted from the cell; the sample of the DNA can completely meet the demands for the researching work of the normal molecule biology and molecule genetics like the PCR analysis, the preparation of a DNA probe, the hybridization of the DNA and the construction of restriction mapping, thus greatly improving the extraction efficiency of DNA and the successful rate is 100 percent.
Description
Technical field
The present invention relates to from cell, extract the method for DNA, particularly a kind of method of extracting cell DNA.
Background technology
Eukaryotic cell has two types of DNA.A kind of is nuclear DNA (nDNA), and it is the research object of being paid much attention at present.Another kind is cytoplasmic DNA (mtDNA), and it is positioned at plastosome, only accounts for 1,000,000 of the total DNA of cell amount/arrive one of percentage.Compare with nDNA, cytoplasmic DNA has different genetic codes and molecular structure.Human cell's matter DNA is a closure, cyclic double chain DNA molecule, and (base pair bp) constitutes, and its nucleotide sequence is clear fully by 16569 base pairs.Studies show that human nerve's degenerative disease, diabetes and old and feeble morbidity and cytoplasmic DNA suddenly change closely related.The focus of malignant cell cytoplasmic DNA molecular structure and expression regulation research becoming gradually research.Obtaining cytoplasmic DNA also is the basis of being engaged in plastosome and chloroplast(id) genetic research.Traditional tenuigenin extracts the DNA method and extracts in two steps, promptly at first plastosome in the cell or chloroplast(id) is separated, and carries out extracting again, its operating process is loaded down with trivial details, need expend a large amount of time and experiment material, gained cytoplasmic DNA quality instability, and limited amount.
Summary of the invention
The object of the present invention is to provide a kind of extracting method of cytoplasmic DNA, directly cytoplasmic DNA is extracted from cell, avoided the extractive in two steps drawback of tradition by this method, and the extraction efficiency height, gained cytoplasmic DNA steady quality.
To achieve these goals, the technical solution adopted in the present invention is:
A kind of extracting method of cytoplasmic DNA comprises that (1) is unicellular from the cell tissue acquisition of human body or animal body, or obtain the step of peripheral leukocytes from the peripheral blood of human body or animal body, and its key is the following step in addition:
(2) distortion and the renaturation of cell, mitochondrial cracking and DNA
In the centrifuge tube that cell is housed, add solution A, add solution A amount by per 10
7Individual cell adds 140～160 μ l meter, and pressure-vaccum is even in ice bath, adds solution B then, and the amount that adds solution B is by per 10
7Individual cell adds 290～310 μ l meter, puts upside down mixing, is placed in the ice bath 5～10 minutes then, adds solution C again, and the amount that adds solution C is by per 10
7Individual cell adds 215～235 μ l meter, and mixing again is placed in the ice bath 20～30 minutes then;
Each solute and concentration thereof are in the above-mentioned solution A: glucose 55mmol/L, EDTANa
22H
2O50mmol/L, Tris-HCl 0.025mmol/L;
Each solute and concentration thereof are in the above-mentioned solution B: SDS 1%, NaOH 0.2mol/L;
Each solute and concentration thereof are in the above-mentioned solution C: potassium ion 3mol/L, acetate ion 5mol/L;
(3) extraction of cytoplasmic DNA
With the solution centrifugal that renaturation finishes, get supernatant liquor, add the saturated phenol of Tris of supernatant liquor 1/2 volume again, the vibration mixing adds the chloroform-primary isoamyl alcohol of supernatant liquor 1/2 volume again, and the volume ratio of chloroform and primary isoamyl alcohol is 24: 1, the mixing that vibrates again, centrifugal then, get the upper strata water;
(4) purifying of cytoplasmic DNA
The Virahol that adds the upper water equal volume of getting, ice bath 25～40 minutes, centrifugal, abandon the upper strata water, centrifugal with 0 ℃ of 65%～75% washing with alcohol precipitation, abandon the supernatant layer, get cytoplasmic DNA after the precipitation drying.
The extracting method of cytoplasmic DNA of the present invention, adopt following process: unicellular or peripheral leukocytes---cell, mitochondrial cracking---DNA distortion and renaturation---DNA extraction and purifying, directly extract cytoplasmic DNA from cell, gained DNA sample can satisfy preparation, the DNA hybridization of conventional molecular biology and molecular genetics such as pcr analysis, dna probe and the needs that limit the research work such as structure of zymogram fully.Improved the extraction efficiency of DNA greatly, and success ratio is 100%.
As preferred embodiment, in step (2), the temperature that adds described solution A is 0 ℃～6 ℃.
As preferred embodiment, in step (2), the temperature that adds described solution B is 15 ℃～25 ℃.
As preferred embodiment, in step (2), the temperature that adds described solution C is 0 ℃～6 ℃.
As preferred embodiment, in step (3), the centrifugal condition of the solution that renaturation finishes is: centrifugal acceleration 9000～11000g, centrifugation time 5～10 minutes, 2 ℃～6 ℃ of centrifuging temperatures.
As preferred embodiment, in step (3), the centrifugal condition behind adding chloroform-primary isoamyl alcohol is: centrifugal acceleration 9000～11000g, centrifugation time 8～15 minutes, 2 ℃～6 ℃ of centrifuging temperatures.
As preferred embodiment, in step (4), the centrifugal condition behind the adding Virahol is: centrifugal acceleration: 14000～16000g, and centrifugation time: 8～15 minutes, centrifuging temperature: 2 ℃～6 ℃.
As preferred embodiment, in step (4), the centrifugal condition behind the adding ethanol is: centrifugal acceleration 14000～16000g, centrifugation time 2～3 minutes, temperature when centrifugal: 2 ℃～6 ℃.
Compare with existing extracting method, the invention has the beneficial effects as follows:
(1) the present invention save time, the success ratio height, the complete cytoplasmic DNA of existing extracting method separation and purification needs 6h just can finish whole experimental implementation, the present invention only needs 4h just can finish, and through the inferior replica tests of 58 examples, success ratio is 100%;
(2) reagent of the present invention's employing is that normal experiment reagent is formulated, and extraction cost is low, and experimental installation is asked not high.
Description of drawings
Fig. 1 is the ultraviolet spectral analysis curve of obtained cytoplasmic DNA sample.
Embodiment
Further the present invention is illustrated below in conjunction with embodiment.
Embodiment 1
(1) preparation of cytoplasmic DNA
Human body pneumonocyte tissue is ground, filter histocyte with metal screen, obtain unicellular, with the TBS liquid (collocation method of TBS liquid: get NaGl 8g, KCl 0.2g, Tris 3g adds deionization distilled water 800ml dissolving, regulate pH value to 7.4 with 2mol/L HCl, distilled water is settled to 1000ml.) cleaning twice, 2000r/min is centrifugal, abandons supernatant, uses the STE liquid (collocation method of STE liquid: get NaCl 5.84g, 0.5mol/L Tris-HCl 20ml, 0.5mol/LEDTANa again
22H
2O 2ml, the dissolving of 800ml deionization distilled water, 2mol/L HCl regulates pH value to 7.4, and distilled water is settled to 1000ml.) wash once, the centrifugal supernatant of abandoning, the cell count in the precipitation is 10
7In cell precipitation, add 4 ℃ of solution A (collocation methods of solution A: get glucose 1g, 0.5mmol/L Tris-HCl 5ml, 0.5mol/L EDTANa
22H
2O 10ml adds deionization distilled water 90ml, and 2mol/L HCl regulates pH value to 8.0, and distilled water is settled to 100ml.) 160 μ l, pressure-vaccum mixing in ice bath, (collocation method of solution B: the 10%SDS 0.2ml with fresh configuration adds 5mol/LNaOH 80ul, adds distilled water 1.72ml, fully mixing to add 25 ℃ of solution B again.) 310 μ l, softly put upside down mixing, cracking in the ice bath, sex change 10 minutes, (collocation method of C solution is: get potassium acetate 29.44g, add distilled water 60ml and fully dissolve, ice acetic acid 11.5ml adds distilled water 28.5ml to add 4 ℃ of solution C again.) 235 μ l, soft mixing, renaturation is 30 minutes in the ice bath.The solution that renaturation finishes is at 10000g, under 4 ℃ of conditions centrifugal 6 minutes, get supernatant liquor, the saturated phenol of Tris that adds supernatant liquor 1/2 volume, gentle vibration 10 minutes, chloroform/the primary isoamyl alcohol that adds former supernatant liquor 1/2 volume again, the volume ratio of chloroform and primary isoamyl alcohol are 24: 1, gentle vibration 5 minutes, then with 10000g, 4 ℃, centrifugal 10 minutes, beat easily the upper strata water, add the Virahol of the upper water equal volume of getting, ice bath 30 minutes, with 15000g centrifugal 10 minutes, the temperature when centrifugal was 4 ℃, abandons the upper strata water, with 0 ℃ of 70% washing with alcohol precipitation, with 15000g centrifugal 2 minutes, the temperature when centrifugal was 4 ℃, abandons supernatant, seasoning precipitation in the air, the gained precipitation is cytoplasmic DNA.
Above-mentioned cytoplasmic DNA also can add the RNA that sneaks in the RNA enzymic digestion operating process in case of necessity, through further concise, can obtain highly purified cytoplasmic DNA.
(2) evaluation of cytoplasmic DNA
Measure A230, A260, A280 value with UV2754 type uv-spectrophotometric instrument, calculate A230/PA260, A260/PA280 ratio, draw nucleic acid ultra-violet absorption spectrum curve, as shown in Figure 1, the result shows, the maximum light absorption value of cytoplasmic DNA sample is at the 260nm place, and the minimum light absorption value presents typical nucleic acid ultra-violet absorption spectrum curve at the 230nm place.
Embodiment 2
Extract anticoagulant heparin peripheric venous blood 1～3ml from human body, get leukocytic cream behind the low-speed centrifugal 5min, clean twice with TBS liquid (collocation method of TBS liquid is with embodiment 1), 2000r/min is centrifugal, abandon supernatant, use STE liquid (collocation method of STE liquid is with embodiment 1) washing more once, the centrifugal supernatant of abandoning, the cell count in the precipitation is 10
7In cell precipitation, add 0 ℃ of solution A (collocation method of solution A is with embodiment 1), 140 μ l, pressure-vaccum mixing in ice bath, add 15 ℃ of solution B (collocation method of solution B is with embodiment 1), 290 μ l again, softly put upside down mixing, cracking in the ice bath, sex change 5 minutes, add 0 ℃ of solution C (collocation method of C solution is with embodiment 1), 215 μ l again, soft mixing, renaturation is 20 minutes in the ice bath.The solution that renaturation finishes is at 9000g, under 2 ℃ of conditions centrifugal 5 minutes, get supernatant liquor, the saturated phenol of Tris that adds supernatant liquor 1/2 volume, gentle vibration 10 minutes, chloroform/the primary isoamyl alcohol that adds former supernatant liquor 1/2 volume again, the volume ratio of chloroform and primary isoamyl alcohol are 24: 1, gentle vibration 5 minutes, then with 9000g, 2 ℃, centrifugal 8 minutes, beat easily the upper strata water, add the Virahol of the upper water equal volume of getting, ice bath 30 minutes, with 15000g centrifugal 10 minutes, the temperature when centrifugal was 4 ℃, abandons the upper strata water, with 0 ℃ of 65% washing with alcohol precipitation, with 14000g centrifugal 3 minutes, the temperature when centrifugal was 2 ℃, abandons supernatant, seasoning precipitation in the air, the gained precipitation is cytoplasmic DNA.
Claims (8)
1. the extracting method of a cytoplasmic DNA comprises that (1) is unicellular from the cell tissue acquisition of human body or animal body, or obtain the step of peripheral leukocytes from the peripheral blood of human body or animal body, it is characterized in that the following step in addition:
(2) distortion and the renaturation of cell, mitochondrial cracking and DNA
In the centrifuge tube that cell is housed, add solution A, add solution A amount by per 10
7Individual cell adds 140～160 μ l meter, and pressure-vaccum is even in ice bath, adds solution B then, and the amount that adds solution B is by per 10
7Individual cell adds 290～310 μ l meter, puts upside down mixing, is placed in the ice bath 5～10 minutes then, adds solution C again, and the amount that adds solution C is by per 10
7Individual cell adds 215～235 μ l meter, and mixing again is placed in the ice bath 20～30 minutes then;
Each solute and concentration thereof are in the above-mentioned solution A: glucose 55mmol/L, EDTANa
2.2H
2O50mmol/L, Tris-HCl 0.025mmol/L;
Each solute and concentration thereof are in the above-mentioned solution B: SDS 1%, NaOH 0.2mol/L;
Each solute and concentration thereof are in the above-mentioned solution C: potassium ion 3mol/L, acetate ion 5mol/L;
(3) extraction of cytoplasmic DNA
With the solution centrifugal that renaturation finishes, get supernatant liquor, add the saturated phenol of Tris of supernatant liquor 1/2 volume again, the vibration mixing adds the chloroform-primary isoamyl alcohol of supernatant liquor 1/2 volume again, and the volume ratio of chloroform and primary isoamyl alcohol is 24: 1, the mixing that vibrates again, centrifugal then, get the upper strata water;
(4) purifying of cytoplasmic DNA
The Virahol that adds the upper water equal volume of getting, ice bath 25～40 minutes, centrifugal, abandon the upper strata water, centrifugal with 0 ℃ of 65%～75% washing with alcohol precipitation, abandon the supernatant layer, get cytoplasmic DNA after the precipitation drying.
2. the extracting method of cytoplasmic DNA according to claim 1, it is characterized in that: in step (2), the temperature that adds described solution A is 0 ℃～6 ℃.
3. the extracting method of cytoplasmic DNA according to claim 1, it is characterized in that: in step (2), the temperature that adds described solution B is 15 ℃～25 ℃.
4. the extracting method of cytoplasmic DNA according to claim 1, it is characterized in that: in step (2), the temperature that adds described solution C is 0 ℃～6 ℃.
5. the extracting method of cytoplasmic DNA according to claim 1, it is characterized in that: in step (3), the centrifugal condition of the solution that renaturation finishes is: centrifugal acceleration 9000～11000g, centrifugation time 5～10 minutes, 2 ℃～6 ℃ of centrifuging temperatures.
6. the extracting method of cytoplasmic DNA according to claim 1 is characterized in that: in step (3), the centrifugal condition that adds behind chloroform-primary isoamyl alcohol is: centrifugal acceleration 9000～11000g, centrifugation time 8～15 minutes, 2 ℃～6 ℃ of centrifuging temperatures.
7. the extracting method of cytoplasmic DNA according to claim 1 is characterized in that: in step (4), the centrifugal condition that adds behind the Virahol is: centrifugal acceleration: 14000～16000g, and centrifugation time: 8～15 minutes, centrifuging temperature: 2 ℃～6 ℃.
8. the extracting method of cytoplasmic DNA according to claim 1 is characterized in that: in step (4), the centrifugal condition that adds behind the ethanol is: centrifugal acceleration 14000～16000g, centrifugation time 2～3 minutes, temperature when centrifugal: 2 ℃～6 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200810070110XA CN101338310A (en) | 2008-08-13 | 2008-08-13 | Process for extracting cytoplasm DNA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200810070110XA CN101338310A (en) | 2008-08-13 | 2008-08-13 | Process for extracting cytoplasm DNA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101338310A true CN101338310A (en) | 2009-01-07 |
Family
ID=40212480
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA200810070110XA Pending CN101338310A (en) | 2008-08-13 | 2008-08-13 | Process for extracting cytoplasm DNA |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101338310A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899432A (en) * | 2010-05-04 | 2010-12-01 | 中国农业科学院油料作物研究所 | Method for extracting high-purity sesame mitochondrion DNA |
| CN102121000B (en) * | 2010-01-07 | 2012-08-22 | 中国农业大学 | Method for extracting mitochondrial DNA of cotton |
| CN106244585A (en) * | 2016-10-11 | 2016-12-21 | 皖南医学院 | A kind of extracting method of simple and efficient oncomelania mitochondrial genome DNA |
-
2008
- 2008-08-13 CN CNA200810070110XA patent/CN101338310A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102121000B (en) * | 2010-01-07 | 2012-08-22 | 中国农业大学 | Method for extracting mitochondrial DNA of cotton |
| CN101899432A (en) * | 2010-05-04 | 2010-12-01 | 中国农业科学院油料作物研究所 | Method for extracting high-purity sesame mitochondrion DNA |
| CN106244585A (en) * | 2016-10-11 | 2016-12-21 | 皖南医学院 | A kind of extracting method of simple and efficient oncomelania mitochondrial genome 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101005924B1 (en) | Nucleic acid extraction apparatus | |
| CN103184268A (en) | Kit for detecting aldehyde dehydrogenase 2 gene polymorphism and amplification method and detection method thereof | |
| WO2016197258A1 (en) | Methods and compositions for cannabis characterization | |
| CN102242212A (en) | HCM (Hypertrophic Cardiomyopathy) genotyping method and kit | |
| CN101338310A (en) | Process for extracting cytoplasm DNA | |
| CN102807976A (en) | Rapid nucleic acid extraction kit and applications thereof | |
| CN101748190B (en) | Method for detecting related loci of type-2 diabetes | |
| CN109536601B (en) | Modeling method of children asthma serum specific miRNAs and decision tree model | |
| CN103374574B (en) | CYP4V2 gene mutant and application thereof | |
| CN106497803B (en) | Fusarium endophytic fungus with huperzine A production function and application thereof | |
| CN101240318A (en) | Kit for detecting senile dementia susceptible inheritance risk | |
| US20200199672A1 (en) | Purification of rna fractions using a hydrophilic polymeric material | |
| CN112941067A (en) | Lysis binding solution for whole blood nucleic acid extraction and kit and application thereof | |
| CN102094002A (en) | Extraction reagent and extraction method of hepatitis B virus DNA | |
| CN102586404B (en) | Kit for specifically detecting genotype of ApoE4 and application of kit | |
| CN104745592A (en) | CYP4V2 gene mutant and application thereof | |
| KR100415726B1 (en) | Sticker for dna isolation and isolation method thereby | |
| CN106222287A (en) | The method of detection ELA2 gene and primer | |
| CN104745594B (en) | CYP4V2 gene mutation body and its application | |
| CN102433322A (en) | Extraction method for honey gene | |
| CN109880822A (en) | A kind of idesia high quality DNA extracting method | |
| CN102329885B (en) | Kit for detecting polymorphism of VKORC1 and CYP2C9 genes | |
| CN106701965A (en) | Mulberry genetic typing method based on single-nucleotide polymorphism markers | |
| WO2020138435A1 (en) | Auxiliary diagnostic method for intraocular malignant lymphoma | |
| CN110699443A (en) | Application of hsa-miR-378i as marker molecule in preparation of tuberculosis diagnosis kit and detection kit thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20090107 |