CN103509786A - Extraction method and applications of inset dry specimen genome - Google Patents
Extraction method and applications of inset dry specimen genome Download PDFInfo
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- CN103509786A CN103509786A CN201310201634.9A CN201310201634A CN103509786A CN 103509786 A CN103509786 A CN 103509786A CN 201310201634 A CN201310201634 A CN 201310201634A CN 103509786 A CN103509786 A CN 103509786A
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Abstract
The invention discloses an extraction method and applications of inset dry specimen genome DNA. The extraction method is based on improvement of saturated NaCl method, and comprises following steps: (1) pretreatment of insect dry specimen; (2) the pretreated material is grinded into powder, and the powder is delivered into a 1.5ml EP tube; (3) solution 1 is added for dissolving so as to remove RNA; (4) digestion liquid 2 is added, the solution is centrifuged, and supernatant is delivered into another EP tube; (5) protein is precipitated; and (6) washing. The extraction method is suitable for DNA extraction of different parts of a plurality of kinds of insect dry specimens such as hymenoptera, odonata, orthoptera and coleoptera; and is high in extraction efficiency. Quality and purity of extracted DNA are capable of meeting requirements of insect molecule system evolution analysis. Basic methods for research and exploration on extraction of insect dry specimen genome DNA at the most appropriate parts with least material amount and least damages are provided. The extraction method is simple and convenient for operation, and applications of the extraction method in labs are convenient.
Description
Technical field
The present invention relates to field of molecular biotechnology, relate in particular to a kind of insect specimen genome DNA extracting method.
Background technology
DNA is the carrier of genetic information, is the main object of molecular biology research.Owing to containing abundant biological information in DNA sequence dna, recorded the history of organic evolution.In active somatic cell, there is complete repair mechanism in DNA, makes DNA intact.But DNA chemical property is extremely unstable, in exsiccata, can spontaneously degrade by hydrolysis or oxygenizement.Exsiccata in sample shop is a huge genetic information storage, modern molecular biology randomly amplified polymorphic DNA (RAPD) technology, restriction fragment length polymorphism (RFLP) technology DNA fingerprint technology and to 12srRNA, 16srRNA, 18SrRNA, the mensuration of the sequences such as ND5, has been widely used in the research of classification of insect.Application DNA sequence dna studies biological phylogeny and evolution laws becomes the focus that current molecular systematics is studied.And the application of these technology, first the most basic problem is the extraction of genomic dna. the developing rapidly of technology makes us be able to disclose on molecular level and utilizes this huge genetic information storehouse possibility that become, and this provides new scientific basis and development opportunity for classification of organisms, phylogeny, ecology and evolution.The material adopting in the extracting method of the genomic dna that many investigators introduce is fresh sample or superfreeze sample (Blin& substantially; Stafford, 1976; Bowtell, 1987; Gross-Bellardd el al., 1972; Kupiec el al., 1987; Wang Wen and Shi Liming, 1993), but in Molecular Phylogeny and Study on Evolution, often there is the monoid of studying, particularly rare monoid is difficult to obtain the problem of fresh material, therefore often need to utilize the exsiccata in the museum of natural history or sample shop to extract genomic dna, but the degraded of longer genomic dna of exsiccata shelf time is also more serious.Therefore, how from exsiccata, to extract the method for genomic dna most important to phylogeny and Study on Evolution.
At present, the main method of extracting insect exsiccata both at home and abroad has: CTAB method, SDS method, Proteinase K method, saturated NaCl method, the imitative extraction process of phenol etc.The imitative extraction process of phenol is the classical way that extracts nucleic acid, and cardinal principle is to utilize the impurity such as nucleic acid, albumen different solubility and redistributing in water and organic phase, is essentially and uses the method for extracting of removing protein after protease K digesting with phenol/chloroform.CTAB method is Murray and Thompson invention, and its principle is that CTAB is a kind of ionic detergent, can make nucleic acid and acidic polysaccharose be precipitated out from the solution of low ionic strength, and meanwhile, the impurity such as protein and neutral polysaccharide are stayed in solution.CTAB method is usually used in the DNA extraction of fresh plant tissue.SDS method, by inventions such as Dellaporta, is utilized the By Direct Pyrolysis cell released dnas such as SDS-Tris-Cl-EDTA, with phenol/chloroform repeatedly after extracting ethanol be settled out DNA.
Tradition DNA extraction method, if SDS method, CTAB method, Proteinase K method are after many experts and scholars improve, only can extract the exsiccata DNA of certain order or some insect.The consumption of the Proteinase K (Kosel& that is directly proportional to the yield of final DNA in Proteinase K method; Grae2ber, 1994; Diaz2Cano& Brady, 1997), in digestive process, add reductive agent (mercaptoethanol or dithiothreitol (DTT) DDT) to seem to improve the yield (Paabo, 1989) of DNA.Generally with ethanol, precipitate DNA.Yet, Kosel& Grae2ber (1994) thinks and extracts in this way specimen dna, and PCR inhibitor can be extracted together with DNA, impact PCR reaction below.Liu Bo etc. (1999) extract and have obtained satisfactory result to the DNA of the exsiccata of oryza chinensis (Oxya) and alcohol-pickled sample respectively by CTAB method.Zhang Shengming etc. utilize SDS method to extract the exsiccata DNA of Coleoptera Scolytidae.Hence one can see that: the above-mentioned various insect exsiccata DNA extraction method scope of application is extremely narrow, only be suitable for the extraction of a certain or a certain insect order exsiccata genomic dna, extraction to the insect exsiccata DNA of other classifications does not obtain good effect, and the imitative extraction process extraction of phenol DNA contains more impurity, for microcomponent with the exsiccata of preserving for many years, these 3 kinds of method extraction effects are poor, can not carry out the follow-up studies such as specific PCR amplification.The insect exsiccata genomic dna of take be both at home and abroad research only concentrate on a few orders such as Hymenoptera, lepidopteran, Coleoptera, and the extracting method of insect exsiccata DNA is different separately.Yet, more than caste, system is huge, etap there are differences, the insect that does not relate to research still can not fast and effeciently be determined extracting method, the huge genetic information storage of containing in insect specimen shop can not utilized, seriously hindered the flow of research of the Molecular Phylogeny and Evolution of insect.Insect molecular systematics research field is in the urgent need to a kind of genome DNA extracting method applicable to a plurality of classification insect exsiccata.
Summary of the invention
The object of this invention is to provide a kind of exsiccata genome DNA extracting method that is applicable to a plurality of order insects.
For realizing goal of the invention, the present invention takes following technical scheme:
An exsiccata genome DNA extracting method, for improved saturated NaCl method, comprises the following steps
(1) obtaining and pre-treatment of insect exsiccata sample, grinds material previously treated to powdery, and grinding product is proceeded to Ep pipe,
(2) add solution (1) to dissolve, except RNA,
(3 add Digestive system (2) digestion, centrifugal, shift supernatant to another Ep pipe,
(4) protein precipitation,
(5) washing,
It is (6) dry,
(7) TE dissolves, and-20 ℃ save backup.
The insect exsiccata sample that step (1) is described for extracting genomic dna, acquisition methods is: from insect specimen chest and foot, head clip muscle 0.03g, in STE damping fluid (0.1mmol/LNaCl, pH8.010mmol/L Tris buffer, pH8.01mmol/L EDTA, dsH2O) soaking at room temperature pre-treatment 5h in is dry on the filter paper of sterilization after taking-up.
The described grinding of step (1) is put into glass rod that Ep pipe adopts sterilizing and is ground at Ep pipe and grind powder that described grinding obtains with dissolving and suspend containing 0.4ml TE.
Described solution 1 consumption of step (2) is 300～400 μ l, described solution (1) is for comprising the 0.4ml TE of 0.1% RNase, the temperature of the described removal of step (3) RNA is 35～37 ℃, and the time is 45～60min, the centrifugal 12000rpm1min of room temperature.
Described Digestive system 1 consumption of step (3) is 300～400 μ l, described Digestive system (2) comprises to be become: 2～8%SDS, the Proteinase K of 100～300 μ g/ml, the PH of described Digestive system (2) is 8.0, the described digestion temperature of step (4) is 25～37 ℃, time is, 16～30h.
The described protein precipitation of step (4) comprises:
1) add the saturated NaCl of 1/4 volume, vortex s or standing 5～8min, the centrifugal 20min of 12000r/min, transfers to another centrifuge tube by supernatant,
2) add isopyknic chloroform, fully mix the centrifugal 15min of rear centrifugal 12000r/min,
3) draw water to another new pipe, after adding the 3M NaAc (pH5.2) of 1/10 volume to mix, add isopyknic Virahol and mix gently, 12000rpm is centrifugal, and 5min is centrifugal, removes supernatant liquor.
The described washing of step (5) is to add 70% ethanol 0.6ml, and the visible cotton-shaped DNA agglomerate of oyster white, fully washs DNA precipitation.
Step (6) is described dry be selected from air seasoning or put dry 3min in 37 ℃ of incubators.
The described TE of step (7) is dissolved as and adds TE (pH8.0) 30～45 μ l dissolving DNAs.
Described 0.1% refers to 100mg/ml, and described 2～8% refer to 2～8g/100ml, 70% described ethanol refer to 70ml straight alcohol with the tri-distilled water constant volume of sterilizing the spirituous solution to 100ml.
TE composition of the present invention is: 10mM Tris-HCl, 0.1Mm EDTA, pH8.0.
The principle of work that the improved NaCl method of the present invention is extracted exsiccata genomic dna is:
Pre-treatment, for extracting the exsiccata material of DNA, is ground material to be extracted to powdery, makes the specimen samples of DNA to be extracted can fully absorb next step solution adding (1).The solution adding (1) containing RNAse, is removed RNA, this than conventional DNA extraction method the preservation of the DNA after extracting step remove again RNA, easier; Afterwards with Digestive system (2) digestion containing SDS and Proteinase K, then by saturated NaCl, chloroform precipitation, remove deproteinize, isopropanol precipitating target dna, dry after 70% washing with alcohol target dna,
Last TE dissolves-20 ℃ of preservations.
2. the evaluation of the target gene group DNA extracting
The multiple classification insect exsiccata genomic dna extracting by present method, can detect its concentration and quality through 0.8% agarose gel electrophoresis and PCR.
PCR detects and can choose copy number mitochondrial COI gene how to using the not equal exsiccata DNA of Hymenoptera of saturated NaCl method extraction to carry out pcr amplification.
3. the purposes of present method
Present method can be applicable to from dragonfly Yan order Odonata, Plecoptera Plecoptera, Orthoptera Orthoptera, Homoptera Homoptera, Hemiptera Hemiptera, Coleoptera Coleoptera, Megaloptera Megaloptera, Diptera Diptera, Trichoptera Trichoptera, Hymenoptera Hymenopteral, lepidopteran Lepidoptera, Ephemeroptera Ephemeroptera, Mantodea Mantodea, Mecoptera Mecoptera, any exsiccata of Neuroptera Neuroptera extracts genomic dna, the DNA content that extraction obtains can meet the requirement of molecular biology subsequent experimental to DNA concentration and quality.The present invention has following advantages and positively effect:
1, the present invention has the caste scope of application widely, can realize the extraction of most of insect order exsiccata DNA.Can extract dragonfly Yan order Odonata, Plecoptera Plecoptera, Orthoptera Orthoptera, Homoptera Homoptera, Hemiptera Hemiptera, Coleoptera Coleoptera, Megaloptera Megaloptera, Diptera Diptera, Trichoptera Trichoptera, Hymenoptera Hymenoptera1, lepidopteran Lepidoptera, Ephemeroptera Ephemeroptera, Mantodea Mantodea, Mecoptera Mecoptera, Neuroptera Neuroptera insect exsiccata DNA.
2, extraction efficiency is high, and the amount of the DNA that carries is higher, and exsiccata DNA quality and purity that successful amplification explanation the present invention of mitochondrial COI gene extracts can be applied to the analysis of insect Molecular Phylogeny and Evolution.
3. can be used in the extraction of insect exsiccata different sites DNA, for research, explore most suitable position, minimum material, minimum damage sample and extract the insect exsiccata genomic dna method that provides the foundation.
4, operation is simple, can carry out in common laboratory, very convenient.
Accompanying drawing explanation
Fig. 1. four kinds of methods are extracted electrophoresis result (the 1. saturated sodium-chloride method of Hymenoptera ichneumon wasp exsiccata genomic dnas; 2.SDS method; 3.CTAB method; 4. Proteinase K method)
Fig. 2. saturated sodium-chloride method is extracted the not equal exsiccata genome dna electrophoresis of Hymenoptera result
(1, Ichneumonidae Ichneumonidae; 2, Apidae Apidae; 3, Vespidae Vespidae; 4, Tenthredinidae Tenthredinidae; 5, Bombidae Bombidae; 6, Formicidae Formicidae; 7, Xylocopidae Xylocopidae.M. molecular weight standard)
Fig. 3. saturated NaCl method is extracted different order insect exsiccata genomic dna results
(1, the Orthoptera locust Acrididae of section; 2, Diptera Tabanidae Acrididaee; 3, Hemiptera Coreidae Coreidae; 4, Homoptera Cicadidae; Cicadidae; 5, the dragonfly Yan Mu Featherlegs Coenagrionidae of section; 6, the Carabidae of Coleoptera Carabidae; 7, the Plecoptera YuanJi Placoptera of section; 8, the Trichoptera phryganeid Phryganeidae of section; 9, the Megaloptera fish fly Corydalidae of section; 10, Hymenoptera Vespidae Vespidae; M. molecular weight standard)
Fig. 4. the Hymenoptera not genomic dna of equal extraction carries out pcr amplification result electrophorogram
1, Vespidae Vespidae; 2, Tenthredinidae Tenthredinidae; 3, Apidae Apidae; 4, Ichneumonidae Ichneumonidae; 5, Formicidae Formicidae; 6, Bombidae Bombidae .M. molecular weight standard)
Embodiment
Below in conjunction with drawings and Examples, describe the present invention in detail:
The improved saturated sodium-chloride method of embodiment 1 is extracted different order insect exsiccata genomic dnas and detection thereof
1.DNA extracts
(1) from insect exsiccata head, chest, foot, obtain for extracting the sample 0.03g of genomic dna, at STE damping fluid (0.1mmol/L NaCl, pH8.0 10mmol/L Tris buffer, pH8.0 1mmol/LEDTA, dsH2O) soaking at room temperature pre-treatment 5h in, dry on the filter paper of sterilization after taking-up
(2) dry material previously treated is put into Ep pipe and is broken to powdery with the glass rod grinding of sterilizing, grinding product is proceeded to Ep pipe,
(3) add solution 1 (the 0.4ml TE that comprises 0.1% RNase) 350 μ l to dissolve, remove RNA;
(4) add Digestive system 2 to comprise 2～8%SDS, the Proteinase K of 100～300 μ g/ml, PH is 8.0) digestion temperature is 25～37 ℃ of digestion 10min, centrifugal, shifts supernatant to another 1.5ml Ep pipe,
(5) protein precipitation:
1) add the saturated NaCl of 1/4 volume, vortex 30s or standing 5～8min, centrifugal 20 min of 12000r/min, transfer to another centrifuge tube by supernatant,
2) add isopyknic chloroform, fully mix the centrifugal 15min of rear centrifugal 12000r/min,
3) draw water to another new pipe, after adding the 3M NaAc (pH5.2) of 1/10 volume to mix, add isopyknic Virahol and mix gently, 12000rpm is centrifugal, and 5min is centrifugal, removes supernatant liquor.
(6) washing: add 70% ethanol 0.6ml, the visible cotton-shaped DNA agglomerate of oyster white, fully washs DNA precipitation
(7) seasoning,
(8) add TE (pH8.0) 30～45 μ l dissolving DNAs ,-20 ℃ save backup.
2. the DNA extracting carries out 1% agarose gel electrophoresis detection
(1.) saturated sodium-chloride method is extracted the not equal exsiccata genomic dna of Hymenoptera agarose gel electrophoresis
(1, Ichneumonidae Ichneumonidae; 2, Apidae Apidae; 3, Vespidae Vespidae; 4, Tenthredinidae Tenthredinidae; 5, Bombidae Bombidae; 6, Formicidae Formicidae; 7, Xylocopidae Xylocopidae.M. molecular weight standard)
Detected result is shown in Fig. 2
(2) saturated NaCl method is extracted different order insect exsiccata genomic dna 1% agarose gel electrophoresis detections
(1, the Orthoptera locust Acrididae of section; 2, Diptera Tabanidae Acrididaee; 3, Hemiptera Coreidae Coreidae; 4, Homoptera Cicadidae; Cicadidae; 5, the dragonfly Yan Mu Featherlegs Coenagrionidae of section; 6, the Carabidae of Coleoptera Carabidae; 7, the Plecoptera YuanJi Placoptera of section; 8, the Trichoptera phryganeid Phryganeidae of section; 9, the Megaloptera fish fly Corydalidae of section; 10, Hymenoptera Vespidae Vespidae; M. molecular weight standard)
Detected result is shown in Fig. 3
(3) insect exsiccata genomic dna saturated sodium-chloride method extraction product P CR amplification electrophoresis ((1, Vespidae Vespidae is seen by the different families of Hymenoptera; 2, Tenthredinidae Tenthredinidae; 3, Apidae Apidae; 4, Ichneumonidae Ichneumonidae; 5, Formicidae Formicidae; 6, Bombidae Bombidae M. molecular weight standard)
Detected result is shown in Fig. 4.
2. four kinds of methods are extracted the electrophoresis detection of Hymenoptera ichneumon wasp exsiccata genomic dnas
(1.) saturated sodium-chloride method; (2.) SDS method; (3.) CTAB method; (4). Proteinase K method
(concrete operation step is according to conventional molecular biology operational guidance)
Detected result is shown in Fig. 1
Above detected result shows, the DNA output that adopts saturated sodium-chloride method to obtain different object insect exsiccata extracting genome DNA can meet the demand of follow-up molecular systematics experiment.
The present invention is not limited to above-mentioned concrete embodiment, and those of ordinary skill in the art is from above-mentioned design, and without performing creative labour, all conversion of having done, within all dropping on protection scope of the present invention.
Claims (9)
1. an insect exsiccata genome DNA extracting method, is improved saturated NaCl method, comprises the following steps:
(1) insect exsiccata sample obtains and pre-treatment
(2) add solution (1) to dissolve, except RNA,
(3) add Digestive system (2) digestion,
(4) protein precipitation,
(5) washing,
It is (6) dry,
(7) TE dissolves, and-20 ℃ save backup;
It is characterized in that, step (1) is described obtains and to be extractedly for extracting the method for the material of DNA, is: described insect exsiccata sample is for for extracting the position of the material of DNA, comprise foot, chest, head,, weight is 0.03g.
2. according to the insect exsiccata genome DNA extracting method of claim 1, it is characterized in that, the pre-treatment of the described insect exsiccata of step (1) is by insect exsiccata sample soaking at room temperature pre-treatment 5h in STE damping fluid, dry on the filter paper of sterilization after taking-up, putting into Ep pipe grinds broken with the glass rod of sterilizing, the powder that described grinding obtains is with dissolving and suspend containing 0.4ml TE, described STE damping fluid consists of 0.1mmol/L NaCl, 10mmol/L Tris bufferpH8.0,1mmol/L EDTA, dsH
2o, the PH of described STE damping fluid is 8.0.
3. according to the insect exsiccata genome DNA extracting method of claim 1, it is characterized in that, described solution 1 consumption of step (2) is 300～400 μ l, described solution (1) is for comprising the 0.4ml TE of 2% RNase, the temperature of the described dissolving of step (2) is 35～37 ℃, time is 45～60min, the centrifugal 12000rpm1min of room temperature after dissolving.
4. according to the insect exsiccata genome DNA extracting method of claim 1, it is characterized in that, the described Digestive system of step (3) (2) consumption is 300～400 μ l, described Digestive system (2) comprises to be become: 2～8%SDS, the Proteinase K of 100～300 μ g/ml, the PH of described Digestive system (2) is 8.0, the described digestion temperature of step (4) is 25～37 ℃, time is, 16～30h.
5. according to the insect exsiccata genome DNA extracting method of claim 1, it is characterized in that, the described protein precipitation of step (4) comprises:
1) add the saturated NaCl of 1/4 volume, vortex 30s or standing 5～8min, the centrifugal 10min of 12000r/min, transfers to another centrifuge tube by supernatant,
2) add isopyknic chloroform, fully mix the centrifugal 10min of rear centrifugal 12000r/min,
3) draw water to another new pipe, after adding the 3M NaAc (pH5.2) of 1/10 volume to mix, add isopyknic Virahol and mix gently, 12000rpm is centrifugal, and 5min is centrifugal, removes supernatant liquor.
6. according to the insect exsiccata genome DNA extracting method of claim 1, it is characterized in that, the described washing of step (5) is to add 70% ethanol 0.6ml, and the visible cotton-shaped DNA agglomerate of oyster white, fully washs DNA precipitation.
7. according to the insect exsiccata genome DNA extracting method of claim 1, it is characterized in that, step (6) is described dry be selected from air seasoning or put dry 3min in 37 ℃ of incubators.
8. according to the insect exsiccata genome DNA extracting method of claim 1, it is characterized in that, the described TE of step (7) is dissolved as and adds TE (pH8.0) 30～45 μ l dissolving DNAs ,-20 ℃ of preservations.
9. insect exsiccata genome DNA extracting method according to claim 1, it is characterized in that, applicable to being selected from dragonfly Yan order Odonata, Plecoptera Plecoptera, Orthoptera Orthoptera, Homoptera Homoptera, Hemiptera Hemiptera, Coleoptera Coleoptera, Megaloptera Megaloptera, Diptera Diptera, Trichoptera Trichoptera, Hymenoptera Hymenoptera1, lepidopteran Lepidoptera, Ephemeroptera Ephemeroptera, Mantodea Mantodea, Mecoptera Mecoptera, the extracting genome DNA of the exsiccata of Neuroptera Neuroptera.
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| CN111635949A (en) * | 2020-07-02 | 2020-09-08 | 大理大学 | DNA barcode standard detection fragment for identifying wasp with yellow veins and application thereof |
| CN111733155A (en) * | 2020-04-27 | 2020-10-02 | 兰州大学 | Method for extracting spider genome DNA |
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| CN105087551A (en) * | 2015-09-18 | 2015-11-25 | 江苏丘陵地区镇江农业科学研究所 | Method for efficient extraction of total RNA in rice planthopper |
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| CN111635949A (en) * | 2020-07-02 | 2020-09-08 | 大理大学 | DNA barcode standard detection fragment for identifying wasp with yellow veins and application thereof |
| CN111635949B (en) * | 2020-07-02 | 2022-10-28 | 大理大学 | DNA barcode standard detection fragment for identifying wasp with yellow veins and application thereof |
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