CN106350510A - Method and kit for quickly extracting genome DNA from forest frog's oviduct, and application thereof - Google Patents
Method and kit for quickly extracting genome DNA from forest frog's oviduct, and application thereof Download PDFInfo
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Abstract
The invention discloses a method for quickly extracting genome DNA from forest frog's oviduct, comprising the steps: (1) taking a forest frog's oviduct sample, adding Chelex-100 and protease K to the forest frog's oviduct sample, carrying out water bath heat insulation first, then terminating at high temperature, carrying out cell cracking, and centrifuging to obtain first supernatant, wherein the Chelex-100 is used for chelating non-nucleic acid organic matters in the forest frog's oviduct sample and inducing protein denaturation to facilitate the protease K to carry out enzymolysis, and the protease K is used for digesting protein in the forest frog's oviduct sample; (2) adding phenol/chloroform/isoamyl alcohol to the first supernatant for removing residual lipoid and protein; and (3) precipitating DNA by using isopropanol or absolute ethyl alcohol. The invention also provides a kit for quickly extracting genome DNA from the forest frog's oviduct, and application of a DNA bar code technology in identification of the forest frog's oviduct. The extraction method disclosed by the invention can realize quick and effective extraction of the genome DNA from dried forest frog's oviduct medicinal materials, and provides a basis for wider application of the DNA bar code technology.
Description
Technical field
The present invention relates to Chinese crude drug genome dna extracts field and in particular to a kind of rapid extraction Oviductus Ranae genome dna
Method, test kit and its application.
Background technology
TCD identification is Study of Traditional Chinese Medicine kind, quality, formulates Chinese medicine standard, finds and expand premise and the basis of medicine source.
Chinese medicine four great tradition authentication method is base identification, character identification, microscopical identification and physical and chemical identification.The theory of conventional identification method
In the analysis of properties and characteristics of taxon, these properties and characteristicses are to be closely related with species growth stage of development and environment to Foundation
Phenotype, affected by subjective and objective factor.And genome dna sequence is the inheritance feature of species, it is biological
Immutable " identity card ", this is animals and plants classification and identification provides essential foundation.
Dna bar code (dna barcoding) technology is by one section of general dna in pcr technology amplification gene group sequence
Fragment, is compared analysis to the general dna fragment of pcr amplification and completes species and fast and accurately identify and identify, in species mirror
Determine aspect and show wide application prospect.At present, carry out identification using coi sequence pair animal medicinal material to have been achieved for extensively
Success.
But Chinese Pharmacopoeia animal drug Oviductus Ranae sample is stale bole sample, fine rich in lymphatic temperament, colloid and half
The materials such as the plain class of dimension, when extracting Oviductus Ranae dna using existing goods dna extracts kit, Oviductus Ranae medicine after adding lysate
Material high level expansion, up to more than 55 times of swelling degree, after water-bath centrifugation, no supernatant or few supernatant is it is impossible to subsequently be breathed out toad
Oily medical material dna extraction step, is unfavorable for the work such as follow-up dna bar code identification.
Therefore, those skilled in the art are devoted to developing a kind of method of rapid extraction Oviductus Ranae genome dna, thus being
The dna bar code identification of this medical material and other application provide good basis.
Content of the invention
When extracting in view of prior art China and Kazakhstan toad oil genome dna, the high level expansion of Oviductus Ranae medical material is it is difficult to quickly have
Effect extracts the defect of genome dna, and the technical problem to be solved is to provide a kind of rapid extraction Oviductus Ranae genome
The method of dna.
For achieving the above object, a first aspect of the present invention provides a kind of side of rapid extraction Oviductus Ranae genome dna
Method, the method comprises the following steps:
1) take Oviductus Ranae sample, add chelex-100 and protease k, first water bath heat preservation, rear high temperature terminates, and carries out cell
Cracking, obtains the first supernatant after centrifugation；Wherein, the non-nucleic acid that chelex-100 is used for chelating in described Oviductus Ranae sample is organic
Thing and make albuminous degeneration be easy to protease k to be digested, protease k is used for digesting the protein in described Oviductus Ranae sample；
2) add phenol/chloroform/isoamyl alcohol in the first supernatant, remove lipid and the protein of residual；
3) isopropanol or dehydrated alcohol is utilized to precipitate dna.
Preferably, the mass volume ratio of chelex-100 is 5%～10%, and the concentration of protease k is 10～20mg/ml.
Further, above-mentioned Oviductus Ranae sample is that Oviductus Ranae medical material is dried.
Further, step 1) further include, after adding chelex-100 and protease k, to this Oviductus Ranae sample
Product are ground pulverizing.
Further, step 1) in water bath heat preservation be 54 DEG C～58 DEG C be incubated 25～35 minutes；High temperature terminates as boiling
It is incubated 5～10 minutes under conditions of boiling, so that membranolysises, and stop the expansion of described Oviductus Ranae sample from absorbing water.
Preferably, water bath heat preservation is 56 DEG C of insulations 30 minutes；Soak is to be incubated 8 minutes under boiling conditions.
Further, add water in the first supernatant and supply volume to 650～750 μ l, be subsequently adding isopyknic step 2)
Described in phenol/chloroform/isoamyl alcohol, and acutely vibrate, after centrifugation, obtain the second supernatant；Wherein said phenol/chloroform/isoamyl alcohol
Volume ratio is phenol: chloroform: isoamyl alcohol=25:24:1.Preferably, add water in the first supernatant and supply volume to 700 μ l
Further, step 2) it is additionally included in described second supernatant, add equal-volume chloroform/isoamyl alcohol, and acutely
Vibration, obtains the 3rd supernatant after centrifugation；The volume ratio of wherein chloroform/isoamyl alcohol is chloroform: isoamyl alcohol=24:1.
A second aspect of the present invention provides a kind of extracts kit of rapid extraction Oviductus Ranae genome dna, this reagent
Box at least includes: the lysate containing chelex-100 and protease k；Impurity removal liquid containing phenol, chloroform and isoamyl alcohol.
Further, in lysate, the mass volume ratio of chelex-100 is 5%～10%, and the concentration of protease k is 10
～20mg/ml.
Further, this extracts kit also includes the sodium acetate of 3mol/l ph 5.2, the te buffer of ph 8.0 and
Grind pestle.
Third aspect present invention provides the method for above-mentioned rapid extraction Oviductus Ranae genome dna or above-mentioned rapid extraction is breathed out
The test kit of toad oil genome dna is identifying the application in Oviductus Ranae using dna bar codes technique.
It is an advantage of the present invention that realizing to quickly effectively the extracting of in Oviductus Ranae medical material genome dna is dried.Wherein,
Using the protein in Proteinase K digestion Oviductus Ranae medical material；Using the good metalloform-selective of chelex-100 and stronger
Adhesion, chelate non-nucleic acid Organic substance, simultaneously by boiling rupture cell membrane, and make non-nucleic acid Organic substance degeneration, it is to avoid
The impact that high level expansion during Oviductus Ranae cracking is extracted to follow-up dna.Afterwards chelex granule can be removed by centrifugation, thus
The impurity such as albumen are separated with dna.The follow-up Impurity removal liquid of joint removes the fatty tissue in sample, further reduces fat
The impact that fat and protein extract to subsequent gene group dna.Using the present invention dna method for extracting it is achieved that to be dried breathe out toad
The quick release of oil difficulty Chinese crude drug sample genome dna and efficiently purifying.The dna being obtained using extracting, enables dna bar shaped
The amplification success rate of code identification technology 100%, this is more widely applied for dna bar codes technique provides good base
Plinth.
Brief description
Fig. 1 is to carry out the electrophoretogram after pcr augmentation detection using the dna extracting acquisition in the embodiment of the present invention 2.Wherein,
Ck is the negative control of pcr.
Specific embodiment
Below with reference to embodiment, the present invention is further described it should be understood that these embodiments are only used as the mesh of illustration
, it is not used in and limit the scope of the invention.
Embodiment 1 is dried Oviductus Ranae genome dna extracting method
Establish the standard method of rapid extraction Oviductus Ranae medical material genome dna, concrete extracting method is:
1. utilize chelex-100 and protease k to be lysate, crack to Oviductus Ranae sample is dried.
2. repeatedly ground using grinding pestle, to no obvious granule, mix.
3. carry out water bath heat preservation, make protease k play Digestion.
4. under boiling conditions be incubated, make cell cracking, simultaneously chelex-100 non-nucleic acid Organic substance is carried out chela and
Under effect, prevent from the imbibition of Oviductus Ranae sample is dried.
5. centrifuging and taking supernatant, and use ddh2O supplements supernatant volume, prevents supernatant very little it is impossible to preferably complete
Become subsequent extraction step.
6. add Impurity removal liquid phenol/chloroform/isoamyl alcohol (25:24:1), centrifugation after vibration mixes obtains supernatant.
7. supernatant adds chloroform/isoamyl alcohol (24:1), and centrifugation after vibration mixes obtains supernatant, thus removing residual
Phenol.
8. supernatant adds sodium acetate to mix, and the dehydrated alcohol adding 2～2.5 times of volumes ice cold mixes, cold preservation, precipitation
dna.This step can also precipitate dna using isopyknic isoamyl alcohol.
9. centrifugation obtains precipitation, adds te buffer or ddh after being dried2O dissolves, that is, obtain genome dna.
Wherein, determine that optimum chelex 100 concentration, optimum are dried Oviductus Ranae sample weighting amount, optimum water using crossing method
Bath temperature retention time, optimum boil temperature retention time.
Specifically, different chelex 100 concentration are compared, 1%, 3%, 5%, 7%, 9%, 10%；
Different Oviductus Ranae sample weighting amounts, 1mg, 5mg, 10mg, 20mg, 30mg；
The different water bath heat preservation time: 10min, 30min, 1h, 2h, 4h, 8h；
Different boiling time: 2min, 4min, 6min, 8min, 10min.
Finally determine optimum chelex 100 concentration be 5%, optimum be dried Oviductus Ranae sample weighting amount be 5mg, optimum water-bath
Temperature retention time is 30min, optimum boils temperature retention time 8min.
Embodiment 2 large sample is verified
In order to verify in embodiment 1 determine be dried Oviductus Ranae genome dna extracting method, reagent composition and concentration general
All over effectiveness, using multiple Oviductus Ranae samples, verified.Particularly as follows:
1) the Oviductus Ranae medical material to different sources (Heilongjiang Province, Jilin Province and Liaoning Province) and the biological system of Chinese medicine are realized
Product examine fixed standard control Oviductus Ranae medical material carry out quick dna extraction.
2) realize carrying out quick dna extraction to the Oviductus Ranae medical material of different commercial specifications (line oil and block oil).
1. take Oviductus Ranae medical material about 5mg, add containing 400 μ l 5%chelex-100's and 4 μ l protease k (20mg/ml)
Lysate.
Wherein, the Oviductus Ranae medical material that is dried of use is respectively as follows:
Draw materials place be Heilungkiang sample: c1, c2, c3；Wherein c1, c2 are line oil, and c3 is block oil；
Draw materials place be Jilin sample: c4, c5, c6；Wherein c4, c5 are line oil, and c6 is block oil；
Draw materials place be Liaoning sample: c7, c8, c9；Wherein c7, c8 are line oil, c9 position block oil；
The standard control Oviductus Ranae medical material of Nat'l Pharmaceutical ＆ Biological Products Control Institute: cb, is powder.
2. repeatedly grind 30sec using grinding pestle, to no obvious granule, mix.
3. 56 DEG C of insulation 30min, shake 10sec.
4. 100 DEG C of insulation 8min, shake 10sec.
5. 14000rpm/min centrifugation 3min, takes supernatant, and uses ddh2O supplements volume to 700 μ l.
6. add equal-volume Impurity removal liquid phenol/chloroform/isoamyl alcohol (25:24:1), turbine mixer acutely vibrates
Mix 30sec, under room temperature, be at full throttle centrifuged 5min.
7. carefully supernatant is transferred to a new centrifuge tube, adds equal-volume chloroform/isoamyl alcohol (24:1), mixed being vortexed
In clutch, acutely vibration mixes 30sec, is at full throttle centrifuged 5min under room temperature.
8. careful sodium acetate supernatant being transferred to a new centrifuge tube, adding the 3mol/l ph 5.2 of 1/10 volume,
Turbine mixer slightly vibrates or flicks centrifugation tube wall with finger and be allowed to several times mix.
9. add dehydrated alcohol or the equal-volume isopropanol of 2～2.5 times of volumes ice cold, turbine mixer vibrates mixed
Even, ice bath 5min, at full throttle it is centrifuged 5min under room temperature.
10. supernatant discarded, adds 70% washing with alcohol of 1ml pre-cooling to precipitate 1～2 time.
11. are deposited in drying in vacuum desiccator or vacuum rotary evaporator, or carefully suck supernatant, will be centrifuged
Pipe is inverted on a piece of paper, and room temperature spontaneously dries.
12. addition 50 μ l te buffer (ph 8.0) or 50 μ l ddh2O dissolves, and preserves at 4 DEG C or -20 DEG C.
The concentration (od value) of dna and purity (od260/280) as shown in table 1:
Table 1 is dried concentration (od value) and the purity (od of Oviductus Ranae sample extraction dna260/280)
| Catalogue number(Cat.No.) | Sample weighting amount (mg) | Od value | od260/od280 |
| c1 | 5.2 | 6.6 | 1.92 |
| c2 | 5.4 | 5.6 | 1.49 |
| c3 | 5.7 | 3.9 | 1.43 |
| c4 | 4.6 | 8.1 | 2.1 |
| c5 | 5.5 | 9.4 | 1.88 |
| c6 | 5.1 | 3.2 | 1.89 |
| c7 | 4.6 | 3.3 | 1.87 |
| c8 | 4.9 | 5.2 | 1.8 |
| c9 | 4.9 | 3.4 | 2.23 |
| cb | 5.6 | 6.2 | 1.81 |
As can be seen here, the dna being obtained using said method and related reagent extracting, is met and is further tested or divide
The requirement of analysis research.
The test kit of embodiment 3 rapid extraction Oviductus Ranae genome dna
This test kit at least includes following component:
1. lysate: containing chelex-100 and protease k, the concentration of chelex-100 is 5%～10%, and protease k is dense
Spend for 10～20mg/ml；
2. Impurity removal liquid: phenol/chloroform/isoamyl alcohol (25:24:1), for remove the fatty tissue in Oviductus Ranae sample and
Albumen etc..
Additionally, the reagent that the routine dna such as te extracts needs can also be included in this test kit.If necessary, can also wrap
Spotting-in muller.
Embodiment 4
Pcr augmentation detection is carried out using the Oviductus Ranae genome dna extracting in embodiment 2.
Prepare pcr system, wherein masterplate is to extract in embodiment 2 to obtain dna.Run pcr amplification program.Pcr product enters
Row dna electrophoresis detection, result is as shown in figure 1, all samples extracting all have amplified band, and band becomes clear, and explanation is taken out
The dna proposing acquisition is applied to and carries out follow-up pcr amplification, provides the foundation for further research.
Claims (10)
1. a kind of method of rapid extraction Oviductus Ranae genome dna is it is characterised in that the method comprising the steps of:
1) take Oviductus Ranae sample, add chelex-100 and protease k, first water bath heat preservation, rear high temperature terminates, and carries out cell and splits
Solution, obtains the first supernatant after centrifugation；Wherein, chelex-100 is used for chelating the non-nucleic acid Organic substance in described Oviductus Ranae sample
Digested with making albuminous degeneration be easy to protease k, protease k is used for digesting the protein in described Oviductus Ranae sample；
2) add phenol/chloroform/isoamyl alcohol in described first supernatant, remove lipid and the protein of residual；
3) isopropanol or dehydrated alcohol is utilized to precipitate dna.
2. the method for rapid extraction Oviductus Ranae genome dna as claimed in claim 1 is it is characterised in that described Oviductus Ranae sample
Product are that Oviductus Ranae medical material is dried.
3. the method for rapid extraction Oviductus Ranae genome dna as claimed in claim 2 is it is characterised in that step 1) further
Including, add chelex-100 and protease k after, using grind pestle described Oviductus Ranae sample is ground pulverize.
4. the method for rapid extraction Oviductus Ranae genome dna as claimed in claim 3 is it is characterised in that step 1) in institute
Stating water bath heat preservation is 54 DEG C～58 DEG C insulations 25～35 minutes；Described high temperature terminates as being incubated 5～10 points under conditions of boiling
Clock, so that membranolysises, and stop the expansion of described Oviductus Ranae sample from absorbing water.
5. the method for rapid extraction Oviductus Ranae genome dna as claimed in claim 3 is it is characterised in that described first supernatant
Liquid adds water and supplies volume to 650～750 μ l, is subsequently adding isopyknic step 2) described in phenol/chloroform/isoamyl alcohol, and acutely
Vibration, obtains the second supernatant after centrifugation；The volume ratio of wherein said phenol/chloroform/isoamyl alcohol is phenol: chloroform: isoamyl alcohol=25:
24:1.
6. the method for rapid extraction Oviductus Ranae genome dna as claimed in claim 5 is it is characterised in that step 2) further
Including in described second supernatant, add equal-volume chloroform/isoamyl alcohol, and acutely vibrate, after centrifugation, obtain the 3rd supernatant；
The volume ratio of wherein said chloroform/isoamyl alcohol is chloroform: isoamyl alcohol=24:1.
7. a kind of test kit of rapid extraction Oviductus Ranae genome dna is it is characterised in that described test kit at least includes: contains
Chelex-100 and the lysate of protease k；Impurity removal liquid containing phenol, chloroform and isoamyl alcohol.
8. the test kit of rapid extraction Oviductus Ranae genome dna as claimed in claim 7 is it is characterised in that described lysate
The mass volume ratio of middle chelex-100 is 5%～10%, and the concentration of protease k is 10～20mg/ml.
9. the test kit of rapid extraction Oviductus Ranae genome dna as claimed in claim 7 is it is characterised in that described extraction tries
Agent box also includes the sodium acetate of 3mol/l ph 5.2, the te buffer of ph 8.0 and grinding pestle.
10. the method or quick as claimed in claim 7 of rapid extraction Oviductus Ranae genome dna as claimed in claim 1
The test kit extracting Oviductus Ranae genome dna is identifying the application in Oviductus Ranae using dna bar codes technique.
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Cited By (1)
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| CN109735601A (en) * | 2019-02-15 | 2019-05-10 | 广州市圣鑫生物科技有限公司 | DNA extraction method and method for paternity test |
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| WO2023131534A1 (en) * | 2022-01-10 | 2023-07-13 | Certest Biotec, S.L. | Method for processing nucleic-acid containing samples |
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| CN1232085A (en) * | 1999-03-31 | 1999-10-20 | 南京师范大学 | PCR determining primer for zaocys dhumnade, tortoise plastron and Oviductus ranae |
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| CN109735601A (en) * | 2019-02-15 | 2019-05-10 | 广州市圣鑫生物科技有限公司 | DNA extraction method and method for paternity test |
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