CN108048449A - A kind of kit for extracting genomic DNA - Google Patents
A kind of kit for extracting genomic DNA Download PDFInfo
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- CN108048449A CN108048449A CN201711388508.3A CN201711388508A CN108048449A CN 108048449 A CN108048449 A CN 108048449A CN 201711388508 A CN201711388508 A CN 201711388508A CN 108048449 A CN108048449 A CN 108048449A
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Abstract
The present invention provides a kind of kit for extracting genomic DNA, reagent 1, reagent 2 and reagents 3.Kit of the present invention enhances the purity and integrality of extracted genomic DNA, improves efficiency of pcr product, agents useful for same is nontoxic, ensures the health of operating personnel, reduces the pollution of environment.Genomic DNA in extractable trace sample realizes the not damaged sampling of detected object, and extraction efficiency is high.
Description
Technical field
Extraction from biological material field more particularly to a kind of examination for extracting genomic DNA the present invention relates to molecular biology
Agent box and extracting method.
Background technology
At present genomic DNA is extracted frequently with the methods of CTAB methods, PTB methods, QIAGEN RNA isolation kits, Low pH extraction with high salts method.
CTAB methods use CTAB (hexadecyltrimethylammonium bromide, cetyl trimethylammonium bromide), it is
A kind of cationic detergent, in the solution of high ionic strength, CTAB forms compound with protein and polysaccharide, and it is heavy to generate
It forms sediment, but compound cannot be formed with nucleic acid and generate precipitation.The supernatant containing nucleic acid is obtained by centrifuging, by adding in chloroform
Organic reagents is waited to be stripped, after removing the impurity such as removing protein, polysaccharide, phenols, nucleic acid can be separated by adding in ethanol precipitation.
CTAB methods need to use the reagents such as beta -mercaptoethanol, chloroform, phenol.But the use of phenol and chloroform can have human body certain harm,
And pollute environment.And extraction probably needs to spend 2-3 days time.PTB methods are needed using EDTA, Proteinase K, PTB, phenol, chloroform
Reagents are waited, operating procedure is extremely cumbersome, and the operating time needs 5-7 days, can be used to phenol, a variety of nuisances of chloroform in operating process
Matter.Low pH extraction with high salts method：It needs using reagents such as EDTA, NaCl, sodium acetate, lauryl sodium sulfate (SDS), chloroform, potassium acetates.
Operating procedure is similar with CTAB methods, but the DNA yield extracted is relatively low and impurity is more.RNA isolation kit：It is easy to use but carry
The yield taken is relatively low, has apparent limitation in the case where sample itself DNA content is low, and cost is higher.It is above this
The deficiency of a little extracting methods and method in itself has been reported that in many documents.
In production, life in people, the protection of timber utilizes into concerned focus with reasonable.With the epoch
Development and people's living standards continue to improve, application of the wooden materials in life are increasingly favored be subject to people, especially
It is that some high-grade rare timber-works also rank among boutique range, becomes the symbol that people pursue quality of the life.Just because of
These treasure rare timber and common wood in price there are huge difference, cause some criminals that some are easily mixed
Confuse and the seeds not being easily distinguishable and timber-work are sold as rare treasures seeds and timber-work, greatly upset red
Wooden market, Archaizing wood market, the normal order in classic furniture market and rare musical instrument market, economically and mentally to consumption
Person brings huge loss and strike.And some forbid felling rare trees also disguised into common wood carry out transport and
Smuggling, this identification work undoubtedly to forestry inspection and customs officer add difficulty.Therefore, in timber processing, utilization, use
In trade activity, timber is carried out it is scientific, rapid and accurate identify and identify be just particularly important with urgently.
Traditional Wood Identification Method requirement assessor have abundant plant gain knowledge with wood structure in terms of specialty know
Know.Usually with traditional wood identification means, the construction and morphological feature of anatomical slice observation timber judge knot so as to draw
Fruit.Since assessor is different to the subjective feeling of specific form feature, even the identification that sometimes professional personnel draw
As a result it is also not quite identical, the especially more similar timber variety of morphological feature.With the development of molecular biology technology, base
It is also applied to because of group DNA analysis in the method for identification timber.And it uses the identification of genomic DNA analysis technology and identifies timber
On condition that sufficient amount and the DNA of high quality are extracted from wood tissue, to meet subsequent PCR amplification, gene sequencing etc.
Molecular biological variety identification method.
Existing extracting method and kit can typically be only used to general plant genome DNA, such as the plants such as blade, stem
The tender tissue of object.Compared to it is fresh and tender and with meristematic capacity plant tissue for, from wood tissue extraction and DNA amplification will
It is difficult very much.Main cause is the following aspects：(1) DNA negligible amounts present in wood tissue, and be usually degraded into small
Segment.(2) there are the tracheary element of heavy wall cause that when milling and handling these hard tissues height can be generated in wood tissue
Temperature, this further damage dna molecule.(3) in timber there are some such as protein, phenols, polysaccharide, tannin and pigment substances,
These substances often influence the activity of archaeal dna polymerase, and interference primer is combined with template, and PCR amplification is caused to fail.(4) timber
It by storage environment influences, if foxiness and fungal contamination can be caused under wet environment, DNA is caused to reduce and pollute.At present
There are no the extracts kits and extracting method for plant xylem genomic DNA.In timber transaction, the cauline leaf of timber is past
It is past all to have extractd.Finished product furniture less has the tender tissues such as cauline leaf naturally, and usually all by processing such as smoked, steaming, extruding.
Therefore the DNA extraction method of existing plant is not appropriate for the DNA extractions of timber and furniture.On the other hand, timber and furniture transaction
It is a quick process of exchange.During the inlet and outlet of timber and furniture, it is also desirable to be completed in a short time the mirror of timber
It is fixed.And existing Method of Plant DNA Extraction usually requires at least 2 days or more, some even wants the time of one week.Therefore it is existing
Extracting method cannot meet the requirement of Rapid identification timber genomic DNA.Existing extracts reagent and method can not extract
DNA in trace sample, in the sample collected for example with existing extracts reagent and extracting method extraction and application invasive methods
DNA, efficiency of pcr product is very poor.
The content of the invention
In order to overcome above-mentioned the problems of the prior art, the present invention provides the kit of extraction genomic DNA, including examination
Agent 1, reagent 2 and reagent 3, wherein the reagent 1 includes following components：Cetyl trimethylammonium bromide (the matter of 0.1%-5%
Measure percentage), PVP (mass percent), the 0.1M- of EDTA, 0.1%-10% of Tris, 10mM-200mM of 10mM-200mM
NaCl, the solvent of 5M is deionized water；Reagent 2 includes following components：The guanidine hydrochloride of 0.1M-6.5M, the ethyl alcohol (body of 1%-50%
Product percentage), the sodium citrate of 0.1M-2M, solvent be deionized water；Reagent 3 includes following components：The guanidine hydrochloride of 0.1M-8M,
Glycine, the solvent of 0.05M-0.2M is deionized water.
Further, the kit further includes cleaning solution.
Further, the kit further includes eluent.
Further, the cleaning solution includes deproteinized cleaning solution and deionization cleaning solution.
Further, the deproteinized cleaning solution includes the guanidine hydrochloride of 1M-6M, the EDTA2Na2H2O of 1mM-50mM,
Solvent is deionized water；The cleaning solution that desalts includes the NaAC3H2O of 20mM-200mM, and solvent is deionized water；Eluent
For deionized water.
Preferably, the reagent includes following components：3.5% cetyl trimethylammonium bromide, 120mM
The EDTA of Tris, 110mM, the NaCl of 6% PVP, 3M, solvent are deionized water；Reagent 2 includes following components：The salt of 2.5M
Sour guanidine, 25% ethyl alcohol, the sodium citrate of 1M, solvent are deionized water；Reagent 3 includes following components：Guanidine hydrochloride, the 0.1M of 4M
Glycine, solvent be deionized water.
The present invention also provides the methods of extraction genomic DNA, comprise the following steps：
(1) sample；
(2) lysate sample cell：The reagent 1 and Proteinase K of 65 DEG C of preheatings are added in, vortex oscillation is uniformly mixed,
65 DEG C of water-bath 0.5h- are stayed overnight；
(3) impurity of non-DNA is precipitated：Mixture in step 2 is cooled to room temperature, adds in reagent 2, is uniformly mixed, ice
Bath；
(4) 3 gained mixture of centrifugation step takes supernatant, adds in carrier RNA and reagent 3, adds in after mixing different
Propyl alcohol, mixing；
(5) DNA in step 4 gained mixture with cmy vector is combined, washs and go by the deproteinized of cleaning solution
Salt washs and the elution step of eluent, obtains the sample genomic dna available for detection；
Wherein, the reagent 1 includes following components：Cetyl trimethylammonium bromide (the quality percentage of 0.1%-5%
Than), the Tris of 10mM-200mM, 10mM-200mM EDTA, the PVP (mass percent) of 0.1%-10%, 0.1M-5M
NaCl, solvent are double distilled deionized water；Reagent 2 includes following components：The guanidine hydrochloride of 0.1M-6.5M, the ethyl alcohol (body of 1%-50%
Product percentage), the sodium citrate of 0.1M-2M, solvent be double distilled deionized water；Reagent 3 includes following components：The salt of 0.1M-8M
Sour guanidine, the glycine of 0.05M-0.2M, solvent are double distilled deionized water.
Further, the cmy vector wherein described in step 4 is selected from nucleic acid extraction purification column or magnetic bead.
Preferably, 65 DEG C of water bath times are 0.5h-2h in the step 2.
Preferably, it is 10min in the step 3 ice bath time.
The beneficial effects of the invention are as follows：Reagent of the present invention and method only need considerably less sample dosage, with regard to that can carry
Get the DNA of needs.Such as using reagent of the present invention and method, it is only the gene in 0.1g samples that can extract sampling quantity
Group DNA.Therefore present invention could apply to minimally invasive sample detection methods, not damaged sampling is carried out to wood sample.It can be to
Solid wood finished product sample after processing extracts, such as available for timber such as various solid wood furnitures, wooden process product, solid wood antiques
The extraction of DNA, this does not destroy the material of furniture etc., and realizes the species identification work of molecular biology level.To treasure
In identification in your species, the present invention plays particularly important effect.The present invention is to traditional CTAB Extraction Methods of Genome
Lysate improved, while improving the concentration of CTAB, add matched reagent, improve the cracking ability of lysate
And the integrality of DNA after purification.It is carried since the water bath time of reagent 1 of the present invention is generally 0.5-2h and can meet DNA
Requirement is taken, therefore extraction time greatly shortens.Agents useful for same of the present invention is nontoxic, ensures the health of operating personnel, avoids duty
Industry disease, it is environmentally safe, it is readily transported.The combination energy of DNA and cmy vector can further be increased by adding in 3 reagent of reagent
Power reduces impurity content, improves product purity, purification efficiency higher.The method of the invention operating procedure is simple, can be significantly
It reduces the operating time, scale operation can be carried out.
Description of the drawings
Fig. 1 is nanmu sample extraction experimental result.
Fig. 2 is different sample dosage test experiments results.
Fig. 3 is sample process time test experimental result.
Fig. 4 be add in reagent 1 after water-bath whether and add in reagent 2 after ice bath whether experimental result.
Fig. 5 is the experimental result for adding in Carrier RNA and reagent 3.
Fig. 6 is to utilize the genomic DNA experimental result in present invention extraction variety classes timber.
Fig. 7 be by the wood-based that Different treatments obtain because extraction experimental result.
Fig. 8 is the gene extraction experimental result of timber different parts.
Fig. 9 is the gene extraction experimental result of different growth years timber.
Figure 10 is the gene extraction experimental result at timber different tissues position.
Figure 11 is reagent selectivity experimental result.
Figure 12 is the experimental result of embodiment 13.
Figure 13 is the experimental result of embodiment 14.
Figure 14 is the experimental result of embodiment 15.
Specific embodiment
The kit of extraction genomic DNA of the present invention includes reagent 1, reagent 2 and reagent 3.Wherein described reagent 1
Including following components：
Cetyl trimethylammonium bromide 0.1%-5% (mass percent)
Tris 10mM-200mM
EDTA 10mM-200mM
PVP 0.1%-10% (mass percent)
NaCl 0.1M-5M
Solvent is deionized water
Reagent 2 includes following components：
Guanidine hydrochloride 0.1M-6.5M
Ethyl alcohol 1%-50% (percent by volume)
Sodium citrate 0.1M-2M
Solvent is deionized water
Reagent 3 includes following components：
Guanidine hydrochloride 0.1M-8M
Glycine 0.05M-0.2M
Solvent is deionized water
Extract genomic DNA reagent include reagent 1, reagent 2 and reagent 3, Proteinase K, Carrier RNA, isopropanol,
Cleaning solution and eluent.
In one embodiment, the deproteinized cleaning solution includes the guanidine hydrochloride of 1M-6M, the EDTA of 1mM-50mM
2Na·2H2O, pH 5.0-8.5, solvent are deionized water.The cleaning solution that desalts includes the NaAC3H of 20mM-200mM2O,
PH is 3.5-7.5, and solvent is deionized water.Eluent is deionized water.The DNA extractions being commercially available on the market at present are with washing
It washs liquid and eluent can also be used for the present invention.
In another embodiment, the pH value of reagent 1 is 4.5-8.5, and the pH value of reagent 2 is 3.3-7.5, the pH of reagent 3
It is worth for 2.5-8.0.
Using kit of the present invention and the method for reagent rapid extraction sample gene group DNA, comprise the following steps：
1. sampling：The sample to be detected is gathered, and obtains the sample for meeting DNA extraction requirements after processing, and by described in
Sample is transferred in centrifuge tube.
2. lysate sample cell：The reagent 1 and Proteinase K of 65 DEG C of preheatings are added in, obturages centrifugation nozzle, vortex shakes
It swings, is uniformly mixed, 65 DEG C of water-bath 0.5h- are stayed overnight.
3. the impurity of the non-DNA of precipitation：Mixture in step 2 is cooled to room temperature, adds in reagent 2, vortex oscillation, mixing
Uniformly, ice bath.
4. step 3 gained mixture is centrifuged, supernatant is taken, carrier RNA and reagent 3 is added in, adds in after mixing
Isopropanol, vortex mixing.
5. the DNA in step 4 gained mixture is combined with cmy vector, wash and desalt by the deproteinized of cleaning solution
Washing and the elution step of eluent, obtain the sample genomic dna available for detection.
Wherein preferred 65 DEG C of water bath times are 0.5h-2h in step 2.The preferred ice bath time is in step 3
10min.Preferably, the centrifugation time is 12,000 × g centrifugations 10min.Preferably, the quantity of isopropanol is about
0.5 times of supernatant volume.
The cmy vector is selected from nucleic acid extraction purification column, magnetic bead etc..
The application of reagent of the present invention and method in the method for column method (preparation tube method) extraction plant genome DNA,
Its method comprises the following steps：
1. weighing vegetable material, liquid nitrogen is added in, the grind into powder in mortar is immediately transferred into centrifuge tube.
2. adding in 65 DEG C of reagents 1 preheated and Proteinase K, lid upper tube cap simultaneously obturaging nozzle, vortex oscillation 30s is mixed
It closes uniformly, 65 DEG C of water-bath 0.5h- are stayed overnight.
3. it is cooled to room temperature, addition reagent 2, ice bath 10min after vortex 1min.
4.12,000 × g centrifuges 10min, takes supernatant into new centrifuge tube, adds in reagent 3 and CarrierRNA, mixes
The isopropanol (such as isopropanol of 360 μ l) of about 0.5 times of supernatant volume, vortex mixing 30s are added in after closing uniformly.
5. prepared by DNA pipes to be placed in new centrifuge tube, take the mixed liquor in step 4 and be transferred to and prepare in pipe, 12,
000 × g centrifuges 1min, abandons filtrate.
6. being put back into pipe is prepared in original centrifuge tube, deproteinized cleaning solution is added in, 12,000 × g centrifugation 1min are abandoned
Filtrate.
7. abandoning filtrate, pipe will be prepared and put back into original 2ml centrifuge tubes, add in the cleaning solution that desalts, 12,000 × g centrifugations
1min abandons filtrate.
8. repeat step 7.
9. it is put back into pipe is prepared in original centrifuge tube, 12,000 × g centrifugations 1min.
10. prepared by DNA pipes to be placed in the centrifuge tube of another cleaning, add deionized water preparing periosteum center, room temperature is quiet
10min is put, 12,000 × g centrifugation 1min eluted dnas obtain genomic DNA.
Reagent of the present invention and method extract the application of plant genome DNA in paramagnetic particle method, and method includes following step
Suddenly：
1. weighing vegetable material, liquid nitrogen is added in, the grind into powder in mortar is immediately transferred into centrifuge tube.
2. adding in the 1 and 20 μ l Proteinase K of reagent of 65 DEG C of preheatings, lid upper tube cap obturages nozzle, whirlpool with sealed membrane
Rotation vibration 30s, is uniformly mixed, 65 DEG C of water-bath 0.5h- are stayed overnight.
3. it is cooled to room temperature, addition reagent 2, ice bath 10min after vortex 1min.
4.12,000 × g centrifuges 10min, takes supernatant into new centrifuge tube, adds in magnetic bead, Carrier RNA, is vortexed
The isopropanol (such as isopropanol of 900 μ l) of about 1.25 times of supernatant volumes, vortex mixing 10min are added in after mixing.
5. centrifuge tube is placed on magnetic frame, is inhaled after supernatant clarification and abandon supernatant.
6. adding deproteinized cleaning solution, be vortexed washing 1min.
7. centrifuge tube is placed on magnetic frame, is inhaled after supernatant clarification and abandon supernatant.
8. adding the cleaning solution that desalts, be vortexed washing 1min.
9. centrifuge tube is placed on magnetic frame, is inhaled after supernatant clarification and abandon supernatant.
10. repeat step 8-9.
11. it is inhaled after of short duration centrifugation and abandons residual liquid, draught cupboard drying.
12. adding deionized water, after mixing, 5min is eluted, obtains genomic DNA.
Embodiment 1 extracts nanmu timber xylem genomic DNA
Extracting the kit of nanmu timber xylem genomic DNA includes reagent 1, reagent 2 and reagent 3.Extract nanmu wood
The reagent of material xylem genomic DNA includes reagent 1, reagent 2, reagent 3, Proteinase K, Carrier RNA, isopropanol, washing
Liquid and eluent.Wherein reagent 1 includes following components：3.5% cetyl trimethylammonium bromide, the Tris of 120mM,
The EDTA of 110mM, the NaCl of 6% PVP, 3M, solvent are deionized water.Reagent 2 includes following components：The guanidine hydrochloride of 2.5M,
25% ethyl alcohol, the sodium citrate of 1M, solvent are deionized water.Reagent 3 includes following components：The guanidine hydrochloride of 4M, 0.1M's is sweet
Propylhomoserin, solvent are deionized water.
The method of extraction and detection nanmu timber genomic DNA comprises the following steps：
1. weighing timber 100mg, liquid nitrogen is added in, the grind into powder in mortar is immediately transferred into 2ml centrifuge tubes.
2. adding in the 1 and 20 μ l Proteinase K of reagent of 900 μ l, 65 DEG C of preheatings, lid upper tube cap simultaneously obturages nozzle, whirlpool
Rotation vibration 30s, is uniformly mixed, 65 DEG C of water-bath 2h.
3. it is cooled to room temperature, 125 μ l reagents 2 of addition, ice bath 10min after vortex 1min.
4.12,000 × g centrifuges 10min, takes 700 μ l of supernatant into new 2ml centrifuge tubes, adds in 20 μ l reagents 3 and 2 μ
L Carrier RNA add in the isopropanol of 360 μ l, vortex mixing 30s after mixing.
5. prepared by DNA pipes to be placed in new 2ml centrifuge tubes, take the mixed liquor in 600 μ l steps 4 and is transferred to preparation pipe
In, 12,000 × g centrifugation 1min abandon filtrate.
6. being put back into pipe is prepared in original 2ml centrifuge tubes, remaining mixed liquor in step 4 is transferred to preparation pipe
In, 12,000 × g centrifugation 1min abandon filtrate.
7. being put back into pipe is prepared in original 2ml centrifuge tubes, 700 μ l deproteinized cleaning solutions, 12,000 × g centrifugations are added in
1min abandons filtrate.
8. put back into pipe is prepared in original 2ml centrifuge tubes, add in 700 μ l and desalt cleaning solution, 12,000 × g centrifugations
1min abandons filtrate.
9. repeat step 8.
10. it is put back into pipe is prepared in original 2ml centrifuge tubes, 12,000 × g centrifugations 1min.
11. prepared by DNA pipes to be placed in the 1.5ml centrifuge tubes of another cleaning, periosteum center plus 100 μ l deionizations are being prepared
Water, is stored at room temperature 10min, and 12,000 × g centrifugation 1min eluted dnas obtain genomic DNA.
The DNA of extraction is subjected to qPCR detections.Wherein amplification condition is：95 DEG C, 2min；95 DEG C of 15s, 55 DEG C of 34s, totally 40
A cycling.QPCR amplifications described in the present embodiment are the ABI 7500RealTime PCR System completions in ABI companies.Also may be used
To be completed in other amplification instruments.The reaction system of qPCR detection is：The system of 20 μ l includes：
Wherein primer and probe is：
Primer UP：5’-CGAAATCGGTAGACGCTACG-3’
Primer Down：5’-TTCCATTGAGTCTCTGCACCT-3’
Prober：5’-GCAATCCTGAGCCAAATCC-3’
Positive control is rice genome.
According to qPCR detection principles, when the DNA of extraction carries out qPCR detections, detection Ct values are less than 35 Xun Huans, then table
It is bright successfully to extract genomic DNA.The Ct values of the present embodiment as shown in Figure 1 are 22.83, this shows to utilize DNA of the present invention
Extracts reagent and extracting method can successfully extract the genomic DNA in 0.1g nanmu xylems.
Contrast experiment of 2 Different Extraction Method of embodiment on extraction Genomic DNA
It weighs dry nanmu wood powder and is utilized respectively reagent component and method of the present invention and establish three experimental groups,
Respectively experimental group 1, experimental group 2 and experimental group 3.And with the CTAB methods of improvement, PTB methods, Low pH extraction with high salts method and Qiagen reagents
Box method extraction timber DNA is control group, and qPCR inspections are carried out respectively after extracting DNA according to experimental group and the respective method of control group
It surveys.Wherein：
The reagent and method of experimental group 1 are referring to embodiment 1；
The reagent component and method of experimental group 2 are with embodiment 1, and the concentration of each component is different from embodiment 1 in reagent.Wherein
Reagent 1 in reagent includes：0.1% cetyl trimethylammonium bromide, the EDTA of the Tris of 15mM, 20mM, 0.2%
The NaCl of PVP, 0.2M, solvent are double distilled deionized water.Reagent 2 includes：The guanidine hydrochloride of 0.5M, 5% ethyl alcohol, the lemon of 0.1M
Sour sodium, solvent are double distilled deionized water.Reagent 3 includes：The guanidine hydrochloride of 0.5M, the glycine of 0.01M, solvent boil off ion to be double
Water.
The reagent component and method of experimental group 3 are with embodiment 1, and the concentration of each component is different from embodiment 1 in reagent.Wherein
Reagent 1 in reagent includes：4.5% cetyl trimethylammonium bromide, the EDTA of the Tris of 180mM, 200mM, 8%
The NaCl of PVP, 4.5M, solvent are double distilled deionized water.Reagent 2 includes：The guanidine hydrochloride of 6M, 45% ethyl alcohol, the lemon of 1.5M
Sour sodium, solvent are double distilled deionized water.Reagent 3 includes：The guanidine hydrochloride of 6M, the glycine of 0.15M, solvent boil off ion to be double
Water.
The CTAB methods of improvement referring to：Wang Guanlin, Fang Hongjun.Plant genetic engineering (second edition), Beijing Science Press,
2002.744。
PTB methods agents useful for same and step are：PTB method main agents include：PTB (bromination N- phenyl acetamides), EDTA, albumen
Enzyme K, phenol chloroform, chloroform isoamyl alcohol, absolute ethyl alcohol, ammonium acetate, TE.PTB method operating procedures include：(1) timber is impregnated with EDTA
Powder 48h makes timber demineralization.(2) after demineralization, Proteinase K and PTB solution are added in 65 degree of water-bath 12h.(3) after water-bath
Add phenol chloroform once.(4) supernatant is taken after centrifuging, chlorination imitates isoamyl alcohol extraction once.(5) step 4 is repeated.(6) nothing is added in
Water-ethanol and ammonium acetate store 12h precipitations DNA in -20 degree refrigerators.(7) centrifuge DNA precipitation washed 2 times with 80% ethyl alcohol
And it is stood overnight in 80% ethyl alcohol.(8) the DNA precipitations for centrifuging totally, TE solution dissolving DNAs are used after dry.
Low pH extraction with high salts method agents useful for same and step are as follows：Low pH extraction with high salts method main agents include：CTAB、5MNaCl、EDTA、
Tris, 35% ethyl alcohol, 70% ethyl alcohol, absolute ethyl alcohol and TE.Low pH extraction with high salts method operating procedure includes：(1) add in wood powder
CTAB extracting solutions, 65 degree of water-bath 2h after mixing.(2) centrifuged after the water-bath plus after 35% ethyl alcohol mixing.(3) supernatant is taken,
Add phenol chloroform once.(4) supernatant is taken after centrifuging, chlorination imitates isoamyl alcohol extraction once.(5) step 4 is repeated.(6) supernatant is taken
Liquid adds the NaCl and absolute ethyl alcohol of 5M, precipitates DNA after mixing, DNA is collected by centrifugation.(7) DNA precipitations are washed with 70% ethyl alcohol.
(8) TE dissolving DNAs are used after drying.
Qiagen RNA isolation kits：It the entitled DNeasy Plant Mini Kit of kit and needs to use
QIAshreder Maxi spin column in DNeasyPlant Maxi Kit.
Table 1：
Testing result is as shown in table 1.It is drawn from contrast and experiment：It can be extracted using reagent of the present invention and method
DNA in trace sample, the time used in extraction is short, and laboratory operating procedures are simply easily mastered.And utilize reagent of the present invention
Seldom consumption of wood is only needed with method, it is possible to which the DNA for extracting sufficient amount is used for subsequent Molecular Detection.
The experiment of 3 wood sample difference sampling quantity of embodiment
Using the nanmu timber of same root drying as material, wood sample sampling quantity is respectively 0.05g, 0.1g, 0.5g, 1.0g
And 2.0g.Using reagent 1, reagent 2 and reagent 3 described in embodiment 1, and using magnetic bead extraction method extraction DNA.The magnetic bead carries
It follows the example of and comprises the following steps：
1. weighing the wood powder of respective amount, liquid nitrogen is added in, the grind into powder in mortar is immediately transferred into 2ml centrifugations
Guan Zhong.
2. adding in the 1 and 20 μ l Proteinase K of reagent of 900 μ l, 65 DEG C of preheatings, lid upper tube cap is obturaged with sealed membrane
Nozzle, vortex oscillation 30s are uniformly mixed, and 65 DEG C of water-bath 0.5h- are stayed overnight.
3. it is cooled to room temperature, 125 μ l reagents 2 of addition, ice bath 10min after vortex 1min.
4.12,000 × g centrifuges 10min, takes 700 μ l of supernatant into new 2ml centrifuge tubes (offer), adds in 20 μ l magnetic
Pearl, 2 μ l Carrier RNA add in the isopropanol of 900 μ l, vortex mixing 10min after vortex mixing.
5. 2ml centrifuge tubes are placed on magnetic frame, are inhaled after supernatant clarification and abandon supernatant.
6. adding 700 μ l deproteinized cleaning solutions, be vortexed washing 1min.
7. 2ml centrifuge tubes are placed on magnetic frame, are inhaled after supernatant clarification and abandon supernatant.
8. 700 μ l is added to desalt cleaning solution, be vortexed washing 1min.
9. 2ml centrifuge tubes are placed on magnetic frame, are inhaled after supernatant clarification and abandon supernatant.
10. repeat step 8-9.
11. it is inhaled after of short duration centrifugation and abandons residual liquid, draught cupboard drying.
12. adding 50-100 μ l deionized waters, vortex elutes 5min after blowing and beating mixing with rifle, obtains genomic DNA.
The DNA of extraction is subjected to qPCR detections, the results are shown in Figure 2.Fig. 2 the result shows that：With the increase of sample size, sheet
The linear increase of yield of the xylem genomic DNA of method extraction, the proportional decline of Ct values of corresponding qPCR, sample applied sample amount
It is strong with result correspondence.
Experiment of the different water bath times of embodiment 4 to extraction effect
Except water bath time is different from embodiment 1 in extracting method, other reagent and methods as described in embodiment 1 are implemented,
Water-bath pyrolysis time used in the present embodiment is respectively 0.5h, 1h, 2h, 4h, 8h, 16h and for 24 hours.
Using different pyrolysis times, the results are shown in Figure 3 by gained qPCR, the results showed that：Water bath time is appropriately extended, it can
The acquisition amount of limited raising genomic DNA.Therefore, when censorship sample quantity is big, shorter water-bath may be employed
Time, to accelerate detection speed.To complete the detection of censorship sample in half day or shorter time.And in the sample size of sampling
When being detected less or for abnormal precious sample, longer water bath time may be employed, improve the efficiency of pcr product of extraction, so as to
Enough genomic DNAs are obtained for follow-up molecular Biological Detection.
Experiment after the addition reagent 1 of embodiment 5 whether water-bath and after addition reagent 2 whether ice bath
In the present embodiment, water-bath is respectively adopted with adding in examination in not water-bath, step 3 except being added in step 2 after reagent 1
Implemented after agent 2 using ice bath and not ice bath, other extracts reagents and method by 1 method of embodiment, complete contrast experiment.Wherein
It compares normal water-bath after adding in reagent 1 to stay overnight, adds in normal ice bath 10min processing after reagent 2.Experimental group 1 is surveyed to try to add in
Not water-bath after agent 1, room temperature processing add in normal ice bath 10min processing after reagent 2.Experimental group 2 is to add in after reagent 1 just
Overnight, adding in reagent 2, ice bath is not handled for ordinary water bath.
The results are shown in Figure 4 by gained qPCR, the results showed that：Reagent 1 is added in, water bath processing is necessary, if not water-bath
Processing, reagent 1 can not just play the role of smudge cells first, secondly Proteinase K just can not digestible protein, final nucleic acid is with regard to nothing
Method releases, therefore experimental result is just displayed without data, illustrates not obtain DNA.Experimental result also shows：Add in reagent
2, ice bath processing is necessary.The effect of ice bath is that the impurity such as albumen is allowed to obtain sufficiently at low ambient temperatures after addition reagent 2
Precipitation is significantly weakened, the removal of impurity is just with reaching deimpurity effect if the sedimentation effect of reagent 2 if ice bath
Not exclusively, the yield of DNA therefore has also been influenced.
Embodiment 6 adds in the experiment of Carrier RNA and reagent 3
In the present embodiment, it is other by real except the adding method difference embodiment 1 of Carrier RNA and reagent 3
The method for applying example 1 is implemented.It tests respectively in the supernatant obtained after centrifugation and adds in the effect of Carrier RNA and reagent 3.
The results are shown in Figure 5 by gained qPCR, wherein, it compares to be not added with Carrier RNA and reagent 3 in supernatant.Experiment
Group 1 is that Carrier RNA, reagent adding 3 are not added in supernatant.Experimental group 2 be in supernatant plus Carrier RNA but not
Reagent adding 3.Experimental group 3 is to add Carrier RNA and reagent 3 simultaneously in supernatant.The result shows that it is individually added into Carrier
RNA or reagent 3 cannot play the efficient combination for promoting DNA and pellosil, only add in Carrier RNA and examination simultaneously
Agent 3 can just make pellosil efficiently combine more DNA.
Embodiment 7 utilizes the DNA in present invention extraction variety classes timber
The timber kinds (be shown in Table 2) different to 15 kinds extract test, and consumption of wood is 100mg.The reagent of extraction
Component and method press together embodiment 1, and a concentration of component is shown in Table 3 in agents useful for same 1, reagent 2 and reagent 3.It is yellow wherein to extract broad-leaved
The extraction of these yellow wingceltis class timber of wingceltis, Ovshinsky yellow wingceltis, nick yellow wingceltis, Barry yellow wingceltis uses the reagent in this row of yellow wingceltis in table 3.Greatly
Red acid branch, Mexico's acid branch, the extracts reagent of Panamanian these sour branch class timber of red acid branch are using the examination in this row of sour branch in table 3
Agent.Africa flower pears, Burma spend these flower pears class timber extracts reagents of pears using the reagent in this row of flower pears in table 3.African acid branch,
These sour branch class timber extracts reagents of Burma's acid branch are using the reagent in this row of sour branch in table 3.Nanmu, gold wingceltis, Indonesia pineapple
Corresponding reagent in table 3 is then respectively adopted in lattice, great Ye red sandalwoods.Extraction gained DNA completes qPCR detections.
Table 2
| Sample number into spectrum | Sample ID | Sample number into spectrum | Sample ID | Sample number into spectrum | Sample ID |
| 1# | Nanmu | 6# | Great Ye red sandalwoods | 11# | Panamanian red acid branch |
| 2# | Broad-leaved yellow wingceltis | 7# | Nick yellow wingceltis | 12# | Africa flower pears |
| 3# | Gold wingceltis | 8# | Big red acid branch | 13# | African acid branch |
| 4# | Ovshinsky yellow wingceltis | 9# | Barry yellow wingceltis | 14# | Burma spends pears |
| 5# | Indonesia's pineapple lattice | 10# | Mexico's acid branch | 15# | Burma's acid branch |
Table 3：
Experimental result such as Fig. 6, the experimental results showed that the method for the invention and reagent can extract variety classes timber
DNA。
The nanmu wood-based that embodiment 8 is obtained by Different treatments because extraction experiment
Using same logs of wood as raw material, and the processing timber handled by different modes is as the present embodiment
Sample.It is extracted by 1 the method for embodiment and reagent and carries out qPCR.
The different processing methods of logs of wood are included being utilized respectively 25 DEG C, 65 DEG C and 105 DEG C three kinds of temperature desiccated woods
Sample；Desiccated wood, the i.e. timber after 25 DEG C of drying at room temperature；Live wood is the timber that makes moist；Extruding is respectively adopted, smokes
Steaming or the wood sample of gas dry-cure.
Experimental result is as shown in Figure 7：The method of the invention and reagent can be with extraction and application distinct methods treated wood
Material DNA.
The gene extraction experiment of 9 timber different parts of embodiment
Using wood of Cunninghamia lanceolata as material, its sapwood and heartwood are taken respectively, and sapwood 1 represents the outermost part of sapwood, 2 table of sapwood
Show interior sap close to the part of heartwood；Heartwood 1 represents the periphery of heartwood, and close to the part of sapwood, heartwood 2 represents heartwood most
Central part.Sapwood：The periphery layer living of trees secondary xylem, positioned at the periphery of trunk, moisture is more compared with heartwood in cell,
Color is shallow, softer, common dark deposit matter in no heartwood.Heartwood：The timber of its nearly central part of perennial trees, without life
Living cells, reserve substance have been not present or have been converted into heartwood substance, and color depth, material is harder and fine and close, and water content is few, no transporting
Infusion and the function of stored nutrient substance, mainly play supporting function to whole plant.Commercial heartwood, which is often referred to pith part, to be had
Notable wood color person.
It is extracted by 1 the method for embodiment and reagent and carries out qPCR.
Experimental result is as shown in Figure 8：The method of the invention and reagent can be with the DNA of extraction and application timber different parts.
The gene extraction experiment of 10 different growth years timber of embodiment
Using wood of Cunninghamia lanceolata as material, tested according to the different years of felling.The China fir is respectively now green wood, life
The wood that timber that timber that the timber cut down after long 1 year, growth are cut down after 2 years, growth are cut down after 3 years, growth are cut down after 5 years
10 years or more material, the timber cut down after growing 10 years, the growth timber cut down.By 1 the method for embodiment and reagent extraction simultaneously
Carry out qPCR.Experimental result is as shown in Figure 9：The method of the invention and reagent can be with the bases of extraction and application timber not growth year
Because of a group DNA.
The gene extraction experiment at 11 timber different tissues position of embodiment
According to 1 the method for embodiment and reagent, it is wooden that Liriodendron xylem, pittosporum tobira xylem, Chinese sweet gum are extracted respectively
Portion, photinia glabra xylem, the genomic DNA of oleander xylem and Liriodendron bast (bark) after qPCR amplifications, carry out
Electrophoresis detection.Electrophoresis result is as shown in Figure 10, and swimming lane 1 is Liriodendron xylem, and swimming lane 2 is pittosporum tobira xylem, and swimming lane 3 is sweetgum
Xylem is set, swimming lane 4 is photinia glabra xylem, and swimming lane 5 is oleander xylem and swimming lane 6 is Liriodendron bast (tree
Skin).The results show that its genome master tape of the timber DNA extracted is clear, it is possible thereby to judge, reagent of the present invention is utilized
It is very good with the integrality of method extraction timber genomic DNA.
12 reagent of embodiment is selectively tested
According to the method described in embodiment 1, the reagent in the selection scheme 1 in table 4-6 and in selection scheme 2 is utilized respectively
Component A, reagent B component and reagent C component, extraction nanmu, Indonesia's pineapple lattice, great Ye red sandalwoods, Mexico's acid branch and African pear flower
In genomic DNA, and complete qPCR measure.
Table 4：
| Reagent A | Selection scheme 1 | Selection scheme 2 |
| Cetyl trimethylammonium bromide | 3.5% | 3.5% |
| Tris | 120mM | 120mM |
| EDTA | 110mM | 110mM |
| PVP | 6% | 6% |
| NaCl | 3M | 3M |
| L-tyrosine | 0 | 0.2% (mass percent) |
Table 5：
| Reagent B | Selection scheme 1 | Selection scheme 2 |
| Guanidine hydrochloride | 2.5M | 2.5M |
| Ethyl alcohol | 25% | 25% |
| Sodium citrate | 0 | 0.8M |
Table 6：
| Reagent C | Selection scheme 1 | Selection scheme 2 |
| Guanidine hydrochloride | 1M | 1M |
| Glycine | 0 | 0.1M |
It can be seen from figure 11 that reagent A-the B of selection scheme 1 has been able to the testing requirement for meeting this experiment, and select
Its is better by reagent A-B in scheme 2, and especially in the extraction of great Ye red sandalwood samples, Ct values improve 1.41.Equally
, on the basis of 2 experimental group 2 of embodiment, increase by 0.1% l-tyrosine in reagent A, increase 0.1M's in reagent B
Sodium citrate increases 0.01M glycine in reagent C.On the basis of 2 experimental group 3 of embodiment, increase by 0.5% in reagent A
L-tyrosine, in reagent B increase 2M sodium citrate, in reagent C increase 0.2M glycine.Its Ct value is also superior to profit
With the Ct values that qPCR is carried out after the reagent extraction DNA of the experimental group 2 and 3 in embodiment 2.
Comparison whether 13 reagent 1 of embodiment uses
In the present embodiment, it is other real as described in Example 1 in addition to the addition of reagent 1 is different from embodiment 1
It applies.Contrast test is adding in reagent 1 and is being added without the effect of reagent 1.
Gained qPCR results are as shown in figure 12, the results showed that：It is necessary to add in reagent 1, and no reagent 1 just can not be to wood
The cell of material sample carries out broken cracking.Therefore experimental result is just displayed without data, illustrates not obtain DNA.
Comparison whether 14 reagent 2 of embodiment uses
In the present embodiment, it is other real as described in Example 1 in addition to the addition of reagent 2 is different from embodiment 1
It applies.Contrast test is adding in reagent 2 and is being added without the effect of reagent 2.
Gained qPCR results are as shown in figure 13, the results showed that：The purification efficiency of timber DNA can be effectively improved by adding in reagent 2.
Add in reagent 2 effectively can carry out precipitation removal to crushing the non-DNA impurity in sample.
Comparison whether 15 reagent 3 of embodiment uses
In the present embodiment, it is other real as described in Example 1 in addition to the addition of reagent 3 is different from embodiment 1
It applies.Contrast test is adding in reagent 3 and is being added without the effect of reagent 3.
Gained qPCR results are as shown in figure 14, the results showed that：Timber DNA and purification efficiency can be effectively improved by adding in reagent 3.
The combination of DNA and cmy vector can be effectively facilitated by adding in reagent 3.
Genomic DNA kit of the present invention, reagent and extracting method can also successfully extract bee milk, insect, silkworm etc.
Genomic DNA in animal.
The above specific embodiment is only in the limited list without prejudice to the present invention under spiritual, it is not excluded that ability
The those skilled in the art in domain the prior art and the present invention combine and generation other specific embodiments.
Claims (6)
1. the kit of genomic DNA is extracted, including reagent 1, reagent 2 and reagent 3, wherein the reagent 1 includes following components：
The cetyl trimethylammonium bromide (mass percent) of 0.1%-5%, Tris, 10mM-200mM of 10mM-200mM
PVP (mass percent), the NaCl of 0.1M-5M, the solvent of EDTA, 0.1%-10% are deionized water；Reagent 2 is included with the following group
Point：The guanidine hydrochloride of 0.1M-6.5M, the ethyl alcohol (percent by volume) of 1%-50%, the sodium citrate of 0.1M-2M, solvent for go from
Sub- water；Reagent 3 includes following components：The guanidine hydrochloride of 0.1M-8M, the glycine of 0.05M-0.2M, solvent are deionized water.
2. kit according to claim 1, which is characterized in that further include cleaning solution.
3. kit according to claim 1, which is characterized in that further include eluent.
4. kit according to claim 3, which is characterized in that the cleaning solution include deproteinized cleaning solution and go from
Seed detergent liquid.
5. kit according to claim 4, which is characterized in that the deproteinized cleaning solution includes the guanidine hydrochloride of 1M-6M,
The EDTA2Na2H of 1mM-50mM2O, solvent are deionized water；The cleaning solution that desalts includes the NaAC of 20mM-200mM
3H2O, solvent are deionized water；Eluent is deionized water.
6. kit according to claim 1, which is characterized in that the reagent 1 includes following components：The ten of 3.5%
Six alkyl trimethyl ammonium bromides, the EDTA of Tris, 110mM of 120mM, the NaCl of 6% PVP, 3M, solvent are deionized water；
Reagent 2 includes following components：The guanidine hydrochloride of 2.5M, 25% ethyl alcohol, the sodium citrate of 1M, solvent are deionized water；Reagent 3 wraps
Include following components：The guanidine hydrochloride of 4M, the glycine of 0.1M, solvent are deionized water.
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| CN107267498A (en) * | 2016-04-08 | 2017-10-20 | 北京爱普拜生物技术有限公司 | DNA kit and method is extracted in a kind of universal material from trace plant |
| CN105754993B (en) * | 2016-04-14 | 2018-06-29 | 中国林业科学研究院木材工业研究所 | A kind of DNA extraction method for seasoned wood |
| CN106399297B (en) * | 2016-09-07 | 2020-02-14 | 贵州茅台酒股份有限公司 | Method for economically and rapidly extracting microbial genome DNA (deoxyribonucleic acid) in fermented grains |
| CN109182328B (en) * | 2018-09-13 | 2022-01-07 | 宋勇 | Mitochondrial DNA extraction kit and method |
| CN110408613B (en) * | 2019-07-31 | 2020-10-27 | 中国林业科学研究院木材工业研究所 | Method for extracting DNA of wood target cell |
| CN113265399A (en) * | 2021-06-18 | 2021-08-17 | 汉远化生医国际科技(北京)有限公司 | Green and rapid animal tissue DNA extraction method |
| CN114369594A (en) * | 2022-02-22 | 2022-04-19 | 上海派森诺生物科技有限公司 | Extraction kit and extraction method for total DNA of microorganisms in excrement |
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| CN103764827A (en) * | 2011-07-04 | 2014-04-30 | 恰根有限公司 | Reagent usable for the isolation and/or purification of nucleic acids |
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