CN106244585B - A kind of extracting method of simple and efficient oncomelania mitochondrial genomes DNA - Google Patents
A kind of extracting method of simple and efficient oncomelania mitochondrial genomes DNA Download PDFInfo
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Abstract
The invention discloses the extracting methods of simple and efficient oncomelania mitochondrial genomes DNA a kind of, comprising the following steps: a) grinding process, b) cracking process, c) precipitation process, d) purification procedures.Compared with prior art, the present invention improving yield by Pintsch process, the digestion of Proteinase K alternating temperature, potassium acetate precipitating, purification step.The present invention mainly uses ultraviolet specrophotometer, agarose gel electrophoresis and PCR detection etc. to carry out quality testing to the mitochondrial genomes DNA of extraction.The result shows that the purity and concentration of the extracted mitochondrial genomes DNA of the present invention have been satisfied with the requirement of the researchs such as population genetic variations, phylogeny, geographical distribution and building gene library.The foundation of this method keeps the compact seashells mitochondrial genomes DNA processes such as extraction oncomelania easier, efficient.
Description
Technical field
The invention belongs to the extracting methods of helminth DNA, particularly belong to the extracting method of oncomelania mitochondrial genomes DNA.
Background technique
Unique intermediate host of the oncomelania as Schistosoma japonicum, distribution are easy by various factors such as water quality, water temperature, weathers
It influences, to generate geographic isolation, the group of Different population even congruence ideal is made to generate multiple kinds of types.Therefore, oncomelania
It hereditary variation and is divided into kind between population between genetic polymorphism status, population between gene intensity of flow, population in order to prevent and treat blood
Emphasis in fluke disease work.Compared with Matrix attachment region, mitochondrial genomes have its unique hereditary capacity, such as maternal something lost
Biography, shortage recombinate and evolutionary rate is fast etc., therefore mtDNA makes a variation to research population genetic and differentiation is of great significance.
When extracting to oncomelania mitochondrial genomes DNA, common extracting method has sucrose density gradient centrifugation, alkaline lysis
Method, Triton method, high salt precipitation method and kit etc., there is operating process complexity, low yields, to human body for these extracting methods
It is toxic, the disadvantages of core DNA pollution is serious, and reagent price is expensive.
Summary of the invention
Technical problem to be solved by the invention is to provide the extraction sides of simple and efficient oncomelania mitochondrial genomes DNA a kind of
Method.
A kind of technical solution of present invention solution technical problem are as follows: extraction of simple and efficient oncomelania mitochondrial genomes DNA
Method, comprising the following steps:
A) grinding process:
By single oncomelania removal spiral case and digestive system tissue, abdominal foot musculature 2-3mg is taken, is put into dismembyator, adds
Enter the homogenate of 50-100 μ l, grinding abdominal foot musculature 1-2 minutes, obtain the homogenate containing musculature, will contain up and down
There is the homogenate of musculature to be transferred in centrifuge tube, add the homogenate of 50-100 μ l, grinds abdominal foot musculature up and down,
It repeats 2-3 times, is ground to abdominal foot muscle into 5~30 μm of particle, finally rinses dismembyator with the homogenate of 100 μ l, form punching
The homogenate containing musculature that grinding obtains for several times is merged with flushing liquor, forms ground homogenate by washing lotion；
B) process is cracked:
The ground homogenate of 300-400 μ l is put into 400-800W micro-wave oven, the high fire of power selection, 6-10 points of heating
Zhong Hou is cooled to room temperature；Heated homogenate liquid is formed, the SDS (final concentration 1%) and 10- of 15-20 μ l are added into heated homogenate liquid
The Proteinase K (300~400 μ g/ml of final concentration) of 15 μ l is uniformly mixed, and is warming up to 60 DEG C, keeps the temperature 1h, is cooled to 37 DEG C, heat preservation
1h；It is warming up to 60 DEG C again, keeps the temperature 1h, is cooled to 37 DEG C, keeps the temperature 1h；Form the homogenate of cracking；
C) precipitation process:
The potassium acetate (PH5.4) of 300 μ l will be added in the homogenate of 300-400 μ l cracking, gently overturns 8~10 up and down
It is secondary, 30-60min is stood at 4 DEG C, in 4 DEG C, 14000r/min, 10-20 minutes min is centrifuged, obtains the 1st supernatant；
It takes the 1st supernatant of 500-600 μ l in centrifuge tube, adds isometric liquor kalii acetici, turn upside down 7~8
It is secondary, 30-60min is stood at 4 DEG C, in 4 DEG C, 12000r/min, 10-20min is centrifuged, obtains the 2nd supernatant；
D) purification procedures:
Take the 2nd supernatant of 900-1000ul in centrifuge tube；Isometric isopropanol, -20 DEG C of refrigeration 30-60min are added
Afterwards, in 4 DEG C, 14000r/min, it is centrifuged 15min, removes supernatant；75% ethyl alcohol (volumetric concentration) of 200-300 μ l is added to be washed
It washs, washes repeatedly 2 times, after ethyl alcohol is air-dried, dissolved with 40-60 μ l distilled water, -20 DEG C save backup.
The pH of the homogenate is 8.0, by following material composition: 25mmol/LTris-HCl, 30mmol/L
EDTANa2, 50mmol/L glucose.
The SDS is 20% lauryl sodium sulfate, and when use is diluted with water to required concentration.
The Proteinase K is 10mg/mL Proteinase K, and when use is diluted with water to required concentration.
The PH of the liquor kalii acetici is 5.4, by material composition below: 3mol/L potassium acetate and 2mol/L acetic acid.
The present invention has the characteristics that following:
Grinding process: since number is uppity to ask there is being homogenized when being ground using traditional homogenizer crush method
Topic, therefore homogenate is added several times, while guaranteeing grinding effect, effectively mitigating leads to small fragment core because being homogenized excessively
The problem of DNA pollution mtDNA.
Cracking process: ground homogenate is placed in micro-wave oven Pintsch process, it is therefore an objective to allow even tissue to disperse, also allow
Cell membrane ruptures to a certain extent, to improve lysis efficiency.On this basis, in Proteinase K water-bath digestion process
Provided with temperature gradient, more mitochondrial genomes DNA are discharged using the sharply change of temperature, obtain the yield of mtDNA
It significantly improves.
Purification procedures: the mitochondrial genomes DNA obtained using potassium acetate purifying eliminates the pollution of core DNA, avoids
The toxic action of the organic matters such as phenol, chloroform decreases the loss of mitochondrial DNA caused by phenol-chloroform extractive process, guarantees
High yield.
A kind of simple and efficient oncomelania mitochondrial genomes DNA extraction method established by the present invention, compared with prior art,
By Pintsch process, the digestion of Proteinase K alternating temperature, potassium acetate precipitating, purification step, yield is improved.The present invention mainly uses purple
Outer spectrophotometer, agarose gel electrophoresis and PCR detection etc. carry out quality testing to the mitochondrial genomes DNA of extraction.Knot
Fruit shows the purity of the extracted mitochondrial genomes DNA of the present invention and concentration has been satisfied with population genetic variations, system is drilled
The requirement of the researchs such as change, geographical distribution and building gene library.The foundation of this method makes to extract the compact seashells mitochondrias such as oncomelania
Genomic DNA process is easier, efficient.
Detailed description of the invention
Fig. 1 is the mitochondrial genomes DNA electrophoresis detection result that embodiment 1 is extracted；
M:DNA Marker-R (0.5kb-14kb) in the figure；The made mitochondrial genomes DNA of A embodiment 1；B sucrose
The made mitochondrial genomes DNA of density-gradient centrifugation method；The made mitochondrial genomes DNA of C high salt precipitation method；
Fig. 2 is that the made mitochondria of embodiment 1 carries out Cox1 gene and IT S sequence fragment amplified production Ago-Gel
Electrophoretogram
M:DNA Marker-J 100+50bp in the figure；DNA；A, B swimming lane is respectively Cox1 gene, ITS gene.
Specific embodiment
Below with reference to embodiment, the present invention is described in detail.
The pH of the homogenate is 8.0, by following material composition: 25mmol/LTris-HCl, 30mmol/L
EDTANa2, 50mmol/L glucose.
The SDS is 20% lauryl sodium sulfate.
The Proteinase K is 10mg/mL Proteinase K.
The PH of the liquor kalii acetici is 5.4, by material composition below: 3mol/L potassium acetate and 2mol/L acetic acid.
For statistical analysis using SPSS17.0 software, mean compares to be examined using t, is that difference has statistics with P < 0.05
Meaning.
Embodiment 1:
A kind of extracting method of simple and efficient oncomelania mitochondrial genomes DNA, comprising the following steps:
A) grinding process:
By single oncomelania removal spiral case and digestive system tissue, abdominal foot musculature 2mg is taken, is put into dismembyator, is added
The homogenate of 50 μ l, grinding abdominal foot musculature 1-2 minutes, obtain the homogenate containing musculature, will contain muscle up and down
The homogenate of tissue is transferred in centrifuge tube, adds the homogenate of 50 μ l, is ground abdominal foot musculature up and down, is repeated 3 times, grinds
Abdominal foot muscle is milled into 5~30 μm of particle, finally rinses dismembyator with the homogenate of 100 μ l, flushing liquor is formed, will grind for several times
It grinds the obtained homogenate containing musculature to merge with flushing liquor, forms ground homogenate；
B) process is cracked:
The ground homogenate of 300 μ l is put into 400W micro-wave oven, the high fire of power selection, heating is after ten minutes, cooling
To room temperature；Heated homogenate liquid is formed, the Proteinase K of the SDS (final concentration 1%) and 10 μ l of 15 μ l are added into heated homogenate liquid
(300 μ g/ml of final concentration) is uniformly mixed, is warming up to 60 DEG C, keeps the temperature 1h, is cooled to 37 DEG C, keeps the temperature 1h；It is warming up to 60 DEG C again,
1h is kept the temperature, is cooled to 37 DEG C, keeps the temperature 1h；Form the homogenate of cracking；
C) precipitation process:
The potassium acetate (PH5.4) of 300 μ l will be added in the homogenate of 300 μ l cracking, it is quiet at gently overturning 8 times, 4 DEG C up and down
30min is set, in 4 DEG C, 14000r/min, 10 minutes min is centrifuged, obtains the 1st supernatant；
It takes the 1st supernatant of 550 μ l in centrifuge tube, adds isometric liquor kalii acetici, it is gently 7 times reverse up and down, 4
30min is stood at DEG C, in 4 DEG C, 12000r/min, is centrifuged 10-20min, is obtained the 2nd supernatant；
D) purification procedures:
Take the 2nd supernatant of 950ul in centrifuge tube；It is added isometric isopropanol, after -20 DEG C of refrigeration 30min, in 4 DEG C,
14000r/min is centrifuged 15min, removes supernatant；200 μ l75% ethyl alcohol (volumetric concentration) are added to be washed, wash repeatedly 2 times,
After ethyl alcohol is air-dried, dissolved with 40 μ l distilled waters, -20 DEG C save backup.
Embodiment 2:
A kind of extracting method of simple and efficient oncomelania mitochondrial genomes DNA, comprising the following steps:
A) grinding process:
By single oncomelania removal spiral case and digestive system tissue, abdominal foot musculature 2.5mg is taken, is put into dismembyator, adds
Enter the homogenate of 80 μ l, grinding abdominal foot musculature 1-2 minutes, obtain the homogenate containing musculature, will contain flesh up and down
The homogenate of meat tissue is transferred in centrifuge tube, adds the homogenate of 80 μ l, is ground abdominal foot musculature up and down, is repeated 2 times,
Abdominal foot muscle is ground into 5~30 μm of particle, finally rinses dismembyator with the homogenate of 100 μ l, forms flushing liquor, it will for several times
It grinds the obtained homogenate containing musculature to merge with flushing liquor, forms ground homogenate；
B) process is cracked:
The ground homogenate of 340 μ l is put into 400W micro-wave oven, the high fire of power selection, heating is after ten minutes, cooling
To room temperature；Heated homogenate liquid is formed, the Proteinase K of the SDS (final concentration 1%) and 15 μ l of 20 μ l are added into heated homogenate liquid
(400 μ g/ml of final concentration) is uniformly mixed, is warming up to 60 DEG C, keeps the temperature 1h, is cooled to 37 DEG C, keeps the temperature 1h；It is warming up to 60 DEG C again,
1h is kept the temperature, is cooled to 37 DEG C, keeps the temperature 1h；Form the homogenate of cracking；
C) precipitation process:
The potassium acetate (PH5.4) of 300 μ l will be added in the homogenate of 350 μ l cracking, it is quiet at gently overturning 8 times, 4 DEG C up and down
30min is set, in 4 DEG C, 14000r/min, 10 minutes min is centrifuged, obtains the 1st supernatant；
It takes the 1st supernatant of 550 μ l in centrifuge tube, adds isometric liquor kalii acetici, it is gently 7 times reverse up and down, 4
30min is stood at DEG C, in 4 DEG C, 12000r/min, is centrifuged 10-20min, is obtained the 2nd supernatant；
D) purification procedures:
Take the 2nd supernatant of 950 μ l in centrifuge tube；It is added isometric isopropanol, after -20 DEG C of refrigeration 30min, in 4 DEG C,
14000r/min is centrifuged 15min, removes supernatant；200 μ l75% ethyl alcohol (volumetric concentration) are added to be washed, wash repeatedly 2 times,
After ethyl alcohol is air-dried, dissolved with 50 μ l distilled waters, -20 DEG C save backup.
Embodiment 3:
A kind of extracting method of simple and efficient oncomelania mitochondrial genomes DNA, comprising the following steps:
A) grinding process:
By single oncomelania removal spiral case and digestive system tissue, abdominal foot musculature 3mg is taken, is put into dismembyator, is added
The homogenate of 100 μ l, grinding abdominal foot musculature 1-2 minutes, obtain the homogenate containing musculature, will contain muscle up and down
The homogenate of tissue is transferred in centrifuge tube, adds the homogenate of 100 μ l, is ground abdominal foot musculature up and down, is repeated 2 times,
Abdominal foot muscle is ground into 5~30 μm of particle, finally rinses dismembyator with the homogenate of 100 μ l, forms flushing liquor, it will for several times
It grinds the obtained homogenate containing musculature to merge with flushing liquor, forms ground homogenate；
B) process is cracked:
The ground homogenate of 400 μ l is put into 400W micro-wave oven, the high fire of power selection, heating is after ten minutes, cooling
To room temperature；Heated homogenate liquid is formed, the Proteinase K of the SDS (final concentration 1%) and 15 μ l of 20 μ l are added into heated homogenate liquid
(340 μ g/ml of final concentration) is uniformly mixed, is warming up to 60 DEG C, keeps the temperature 1h, is cooled to 37 DEG C, keeps the temperature 1h；It is warming up to 60 DEG C again,
1h is kept the temperature, is cooled to 37 DEG C, keeps the temperature 1h；Form the homogenate of cracking；
C) precipitation process:
The potassium acetate (PH5.4) of 300 μ l will be added in the homogenate of 400 μ l cracking, it is quiet at gently overturning 8 times, 4 DEG C up and down
30min is set, in 4 DEG C, 14000r/min, 10 minutes min is centrifuged, obtains the 1st supernatant；
It takes the 1st supernatant of 550 μ l in centrifuge tube, adds isometric liquor kalii acetici, it is gently 7 times reverse up and down, 4
30min is stood at DEG C, in 4 DEG C, 12000r/min, is centrifuged 10-20min, is obtained the 2nd supernatant；
D) purification procedures:
Take the 2nd supernatant of 950 μ l in centrifuge tube；It is added isometric isopropanol, after -20 DEG C of refrigeration 30min, in 4 DEG C,
14000r/min is centrifuged 15min, removes supernatant；200 μ l75% ethyl alcohol (volumetric concentration) are added to be washed, wash repeatedly 2 times,
After ethyl alcohol is air-dried, dissolved with 60 μ l distilled waters, -20 DEG C save backup.
Embodiment 4:
Ultraviolet spectrophotometry is the common method of current measurement DNA concentration and purity, and the result of measurement is accurate.When
When A260/A280 ratio is between 1.8~2.0 out, show that mtDNA mass is preferable；When less than 1.8, illustrate there is protein
Pollution shows there is RNA pollution when being greater than 2.0；
By the made DNA of embodiment 1 and the common extracting method sucrose density gradient centrifugation of mesh first two and with high salt heavy
Shallow lake method compares.Wherein sucrose density gradient centrifugation is according to (Tamura K, Aotsuka T.Rapid isolation
method of animal mitochondrial DNA by the alkaline lysis procedure[J]
.Biochemical Genetics, 1988,26 (11-12): 815-819.) it extracts, high salt precipitation method is according to (Zheng Runling, Li Jia
Happy, Niu Donghong hangs comparison [J] molecular science journal of razor clam mitochondrial DNA extracting method: Chinese and English version, 2008,24 (5):
312-315.) extract mitochondrial genomes DNA.
MtDNA sample obtained is diluted by 1:100, measures light absorption angle value with UV detector.Its result is such as
Shown in table 1:
The result shows that the OD value of three kinds of extracting methods is between 1.8~2.0, no significant difference (F=
0.008,1.076, P > 0.05)；The yield of embodiment 1 has system better than sucrose density gradient centrifugation and high salt precipitation method, difference
Meter learns meaning (F=24.686,16.428, P < 0.05),
Embodiment 5:
Agarose gel electrophoresis method forms complex compound between utilizing ethidium bromide (EB) embeddable DNA molecular base-pair, through purple
After outside line irradiation, mtDNA concentration and purity are judged roughly by the brightness of band；
1% Ago-Gel is prepared, the mitochondrial genomes DNA sample of 3 μ l and the loading Buffer of 3ul are taken
(30mM (w/v) Xylene Cyanol of EDTA, 50% (v/v) Glycerol, 0.25% FF, 0.25% (w/v)
Bromophenol Blue) it is mixed, 80V 40min, gel imager is imaged and takes pictures.
Its result as shown in Figure 1, three kinds of extracting methods extract mitochondrial DNA stripe size about 15kb, wherein implementing
The extraction effect of example 1 is best, and band is brighter and loading wells is nearby without obvious band, shows that core DNA and protein contamination are less.
Embodiment 6:
The ITS sequence of the extracted mtDNA Cox1 gene of embodiment 1 and karyogene is expanded, core is detected the presence of
The pollution of DNA.
It expands Cox1 gene primer and ITS sequence primer and designs (table 2) referring to having document, to measure mitochondrial DNA expansion
Increase and core DNA pollution situation.PCR reaction system includes 4 μ l of template, 17 μ l ultrapure waters, 25 μ l Mix (2 × Taq PCR
MasterMix), primers F, each 2 μ L of R (1mmol/L).PCR reaction condition: 94 DEG C of 5min of Cox1；94 DEG C of 30s, 51 DEG C of 50s, 72
DEG C 1min is recycled 34 times；72 DEG C of extension 10min；ITS annealing temperature is 50 DEG C, and other conditions are identical as Cox1, amplified production row
1% agarose gel electrophoresis is analyzed, and the brightness of each band is analyzed under ultraviolet light.
The primer and sequence of amplification Cox1, ITS gene of table 2
The PCR amplification result of mitochondrial genomes DNA is as shown in Fig. 2, Cox1 gene amplification product map is 700bp or so
Segment serves the sequencing of marine growth Engineering Co., Ltd, sequencing result is inquired through GenBank, discovery and oncomelania hupensis mtDNA
Cox1 genetic homology is determined as chondriogen 99%.And ITS is without obvious band, the results showed that karyogene content is less.
Claims (1)
1. a kind of extracting method of oncomelania mitochondrial genomes DNA, comprising the following steps: a) grinding process, b) crack process, c)
Precipitation process, d) purification procedures；
A) the grinding process:
By single oncomelania removal spiral case and digestive system tissue, abdominal foot musculature 2-3mg is taken, is put into dismembyator, 50- is added
The homogenate of 100 μ l, grinding abdominal foot musculature 1-2 minutes, obtain the homogenate containing musculature, will contain muscle up and down
The homogenate of tissue is transferred in centrifuge tube, adds the homogenate of 50-100 μ l, grinds abdominal foot musculature up and down, repeats 2-
3 times, abdominal foot muscle is ground into 5~30 μm of particle, finally rinses dismembyator with the homogenate of 100 μ l, forms flushing liquor, it will
The homogenate containing musculature that grinding obtains for several times merges with flushing liquor, forms ground homogenate；
The b) cracking process:
The ground homogenate of 300-400 μ l is put into 400-800W micro-wave oven, the high fire of power selection heats 6-10 minutes
Afterwards, it is cooled to room temperature, forms heated homogenate liquid；The SDS of 15-20 μ l is added into heated homogenate liquid, makes the final concentration 1% of SDS,
With the Proteinase K of 10-15 μ l, make 300~400 μ g/ml of final concentration of Proteinase K, be uniformly mixed, be warming up to 60 DEG C, keeps the temperature 1h,
37 DEG C are cooled to, 1h is kept the temperature；It is warming up to 60 DEG C again, keeps the temperature 1h, is cooled to 37 DEG C, keeps the temperature 1h, forms the homogenate of cracking；
The c) precipitation process:
The potassium acetate of the PH5.4 of 300 μ l will be added in the homogenate of 300-400 μ l cracking, it is quiet at turning upside down 8~10 times, 4 DEG C
30-60min is set, in 4 DEG C, 14000r/min, 10-20min is centrifuged, obtains the 1st supernatant；
It takes the 1st supernatant of 500-600 μ l in centrifuge tube, adds isometric liquor kalii acetici, turn upside down 7~8 times, 4
30-60min is stood at DEG C, in 4 DEG C, 12000r/min, is centrifuged 10-20min, is obtained the 2nd supernatant；
The d) purification procedures:
Take the 2nd supernatant of 900-1000ul in centrifuge tube；It is added isometric isopropanol, after -20 DEG C of refrigeration 30-60min, in
4 DEG C, 14000r/min, it is centrifuged 15min, removes supernatant；The ethyl alcohol that 200-300 μ l75% volumetric concentration is added is washed, and is repeated
Washing 2 times after air-drying ethyl alcohol, is dissolved, -20 DEG C save backup with 40-60 μ l distilled water；
The pH of the homogenate is 8.0, by following material composition: 25mmol/L Tris-HCl, 30mmol/L EDTANa2With
50mmol/L glucose.
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