CN104371998A - DNA extracting method for bitter gourd hybrid purity molecular identification - Google Patents
DNA extracting method for bitter gourd hybrid purity molecular identification Download PDFInfo
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- CN104371998A CN104371998A CN201410683660.4A CN201410683660A CN104371998A CN 104371998 A CN104371998 A CN 104371998A CN 201410683660 A CN201410683660 A CN 201410683660A CN 104371998 A CN104371998 A CN 104371998A
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Abstract
The invention provides a DNA extracting method for bitter gourd hybrid purity molecular identification. The method comprises the following steps: on the basis of extracting genome DNA through an improved CTAB method, respectively feeding a large number of bitter gourd tender leaves from different plants required for detecting in 1.5ml centrifugal tubes, placing the centrifugal tubes in a refrigerator at -80 DEG C for more than 3 hours, taking out, grinding about 5s by using a cleaned grinding pestle, quickly adding 2*CTAB extracting buffer solution, and then suspending through chloroform/isoamylol, washing and precipitating through isopropanol and 70% ethanol to obtain a large number of to-be-detected sample bitter gourd genome DNA of different plants. The problem that the step of extracting the bitter gourd genome DNA by using the traditional CTAB method is excessively fussy is solved; and meanwhile, the different to-be-detected bitter gourd genome DNA obtained through extracting is basically the same in concentration, and can be completely suitable for molecular identification of RAPD, SRAP and ISSR of bitter gourd hybrid purity in quality and completeness.
Description
Technical field
The invention belongs to technical field of molecular biology, more specifically relate to a kind of DNA extraction method for balsam pear purity of hybrid Molecular Identification.
Background technology
In recent years, report RAPD, SRAP and ISSR equimolecular mark is had to apply to balsam pear purity of hybrid Molecular Identification, but the individual specimen amount needed due to Molecular Identification balsam pear purity of hybrid is large, and usual way liquid nitrogen grinding tender leaf extraction balsam pear D N A step is too loaded down with trivial details.Therefore, develop easy, feasible fast balsam pear D N A extracting method, just seem very important to obtain desirable target DNA for Molecular Identification.Though more existing conventional D N A extraction methods are through updating and simplifying, step is still more, and proceeds in centrifuge tube after mostly needing liquid nitrogen grinding again.Certainly the method also having report very easy extracts some DNA of plants such as corn, but these too easy methods cannot obtain desirable balsam pear object D N A, therefore can not be used for the qualification of balsam pear cross-fertilize seed RAPD, SRAP and ISSR equimolecular.
And the present invention adopts a kind of without the need to liquid nitrogen grinding method extraction balsam pear tender leaf DNA, greatly simplifie the extraction step of balsam pear D N A, according to said method, everyone can complete about 144 balsam pear sample DNAs and extract in one day.
Summary of the invention
The object of the present invention is to provide the DNA extraction method for balsam pear purity of hybrid Molecular Identification, solve traditional CTAB method and extract the too loaded down with trivial details step of balsam pear tender leaf DNA (adopting stone roller alms bowl and liquid nitrogen grinding generally can only extract about 48 sample DNAs in everyone day).The invention provides one utilizes improved method of CTAB to extract balsam pear tender leaf genome DNA (everyone can carry about 144 samples for a day), the method is easy, suitability is strong, takes a firm foundation for balsam pear goal gene group STb gene carries out the Purities such as RAPD, SRAP and ISSR.
Improved method of CTAB is adopted to extract not homophyletic cross-fertilize seed balsam pear tender leaf genome DNA, but without the need to putting into mortar liquid nitrogen grinding, put into-80 DEG C of more than refrigerator 3h, rear pestle grinding about the 5s that mills taken out with cleaning, add rapidly 2 × CTAB Extraction buffer, chloroform again/primary isoamyl alcohol suspends, and Virahol and 70% washing with alcohol precipitation, finally obtain a large amount of not homophyletic detected sample balsam pear genomic dna.
The invention provides the total genome DNA Perfected process of a kind of extraction balsam pear tender leaf, specifically comprise:
(1) in the different centrifuge tube of 1.5 ml, putting into each 0.1-0.3g of different balsam pear tender leaf sample DNA to be detected, the centrifuge tube that balsam pear tender leaf is housed being placed on flat board, without the need to directly putting into-80 DEG C of freezing 3-5h of refrigerator after lid pipe;
(2) in refrigerator, take out the centrifuge tube that balsam pear tender leaf is housed successively, grinding 5-7 second fast with cleaning the pestle of milling after drying, adding 2 × CTAB(cetyl trimethylammonium bromide that 500 μ l are preheated to 60 DEG C) Extraction buffer, fully mix;
(3) after all samples is milled and added 2 × CTAB Extraction buffer, put into 65 DEG C of water-bath 12-25 min together, period puts upside down mixing 1 time, takes out after water-bath, adds isopyknic chloroform isoamyl alcohol mixed solution in often managing, centrifugal 10 min of 12000r/min;
(4) supernatant liquor 300 μ l is got to another centrifuge tube, add the pH5.2 of equal-volume Virahol and 1/10 volume, 3 mol/L NaAc(sodium-acetates), place 10 min, centrifugal 5 min of 12000 r/min under 4 DEG C of conditions, white precipitate DNA concentration be 70% washing with alcohol once after, air-dry in super clean bench;
(5) target DNA after air-dry is dissolved in 50 μ LTE solution, adds 10 g/L, 0.5 μ LRNase, and in 37 DEG C of incubation 15 min; Namely save backup for Molecular Identification or in-2O DEG C.
In step (3), in chloroform isoamyl alcohol mixed solution, each volume components ratio is 24:1.
The method, without the need to adding liquid nitrogen grinding balsam pear tender leaf DNA with stone roller alms bowl, eliminates and adds with a large amount of alms bowl that grinds the tedious steps proceeding to centrifuge tube after liquid nitrogen grinds balsam pear tender leaf again, save the plenty of time.Simultaneously often the balsam pear DNA concentration extracted of pipe is basically identical and quality and integrity are all better, is suitable for very much the Molecular Identification that RAPD, SRAP and ISSR equimolecular mark carries out a large amount of balsam pear cross-fertilize seed sample purity.
Accompanying drawing explanation
Fig. 1: different sample DNA TaKaRa (Code:D520A) the DNA Marker obtained in contrast.From agarose gel electrophoresis image schematic diagram 1, the different sample target DNA concentration obtained are basically identical, all between 50ng-150ng, target DNA purity and integrity are comparatively general, but do not affect next step and carry out the Molecular Identification that RAPD, SRAP and ISSR equimolecular mark carries out a large amount of balsam pear cross-fertilize seed sample purity.
Fig. 2: the DNA application RAPD technology that aforesaid method extracts and primer CCTTGACGCA carry out new emerald green balsam pear Hybrid seed purity test, obtain the false cross-fertilize seed that a strain has maternal characteristic bands, this result fits like a glove in field planting purity detecting result, illustrates that the DNA that this method is extracted can be used for new emerald green balsam pear Hybrid seed purity test; Wherein 1 represents that 1 strain has the false cross-fertilize seed of maternal characteristic bands.
Fig. 3: the DNA application SRAP technology that aforesaid method extracts and primer: TGAGTCCAAACCGGAAG; GACTGCGTACGAATTTGA carries out as beautiful No. 5 balsam pear Hybrid seed purity tests, obtain the false cross-fertilize seed that two strains have maternal characteristic bands, this result fits like a glove in field planting purity detecting result, illustrates that DNA that this method extracts can by the Molecular Identification with such as beautiful No. 5 balsam pear purity of hybrid; Wherein 1 represents that 2 strains have the false cross-fertilize seed of maternal characteristic bands.
Fig. 4: the DNA application ISSR technology that aforesaid method extracts and primer: ACACACACACACACACCTG carry out as beautiful No. 41 balsam pear Hybrid seed purity tests, obtain the false cross-fertilize seed that two strains have maternal characteristic bands, this result fits like a glove in field planting purity detecting result, illustrate that DNA that this method is extracted can be used for the Molecular Identification as beautiful No. 41 balsam pear purity of hybrid, wherein 1 represents that 2 strains have the false cross-fertilize seed of maternal characteristic bands.
Embodiment
embodiment 1
(1) in the different centrifuge tube of 1.5 ml, putting into each 0.2g of different balsam pear tender leaf sample DNA to be detected, the centrifuge tube that balsam pear tender leaf is housed being placed on flat board, without the need to directly putting into-80 DEG C of freezing 3h of refrigerator after lid pipe;
(2) in refrigerator, take out the centrifuge tube that balsam pear tender leaf is housed successively, grinding 5 seconds fast with cleaning the pestle of milling after drying, adding 2 × CTAB Extraction buffer that 500 μ l are preheated to 60 DEG C, fully mixing;
(3) after all samples is milled and added 2 × CTAB Extraction buffer, put into 65 DEG C of water-bath 15 min together, period puts upside down mixing 1 time, takes out after water-bath, adds isopyknic chloroform isoamyl alcohol mixed solution in often managing, centrifugal 10 min of 12000r/min;
(4) supernatant liquor 300 μ L is got to another centrifuge tube, add the pH5.2 of equal-volume Virahol and 1/10 volume, 3 mol/L NaAc(sodium-acetates), place 10 min, centrifugal 5 min of 12000 r/min under 4 DEG C of conditions, white precipitate DNA concentration be 70% washing with alcohol once after, air-dry in super clean bench;
(5) target DNA after air-dry is dissolved in 50 μ LTE solution, adds 10 g/L, 0.5 μ LRNase, and in 37 DEG C of incubation 15 min; Namely save backup for Molecular Identification or in-2O DEG C.
embodiment 2: the inventive method extracts the balsam pear DNA concentration and purity that obtain
The different sample DNAs obtained respectively get 5 μ L, get 5 μ L TaKaRa (Code:D520A) DNA Marker in contrast, with the Agarose of 1%, voltage is about 10V/cm simultaneously, EB dyes about 5min after electrophoresis about 1h, takes pictures after observing under ultraviolet lamp.
From electrophoretic image schematic diagram 1, the different sample target DNA concentration obtained are basically identical, all between 50ng-150ng, target DNA purity and integrity are comparatively general, but do not affect next step and carry out the Molecular Identification that RAPD, SRAP and ISSR equimolecular mark carries out a large amount of balsam pear cross-fertilize seed sample purity.
embodiment 3: RAPD, SRAP and ISSR equimolecular qualification result of different balsam pear purity of hybrid
(1) aforesaid method extract DNA application RAPD technology and primer CCTTGACGCA carry out new emerald green balsam pear Hybrid seed purity test, obtain the false cross-fertilize seed (see figure 2) that a strain has maternal characteristic bands, this result fits like a glove in field planting purity detecting result, illustrates that the DNA that this method is extracted can in order to the Molecular Identification of new emerald green balsam pear purity of hybrid;
(2) aforesaid method extract DNA application SRAP technology and primer: TGAGTCCAAACCGGAAG; GACTGCGTACGAATTTGA carries out as beautiful No. 5 balsam pear Hybrid seed purity tests, obtain the false cross-fertilize seed (see figure 3) that two strains have maternal characteristic bands, this result fits like a glove in field planting purity detecting result, illustrates that DNA that this method extracts can in order to the Molecular Identification of such as beautiful No. 5 balsam pear purity of hybrid;
(3) aforesaid method extract DNA application ISSR technology and primer: ACACACACACACACACCTG carry out as jade No. 41 balsam pear Hybrid seed purity tests, obtain the false cross-fertilize seed (see figure 4) that two strains have maternal characteristic bands, this result fits like a glove in field planting purity detecting result, illustrates that DNA that this method extracts can in order to the Molecular Identification of such as beautiful No. 41 balsam pear purity of hybrid.
SEQUENCE LISTING
<110> Sugarcane Inst., Fujian Provincial Academy of Agricultural Science
<120> is used for the DNA extraction method of balsam pear purity of hybrid Molecular Identification
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 10
<212> DNA
<213> artificial sequence
<400> 1
ccttgacgca 10
<210> 2
<211> 17
<212> DNA
<213> artificial sequence
<400> 2
tgagtccaaa ccggaag 17
<210> 3
<211> 18
<212> DNA
<213> artificial sequence
<400> 3
gactgcgtac gaatttga 18
<210> 4
<211> 19
<212> DNA
<213> artificial sequence
<400> 4
acacacacac acacacctg 19
Claims (3)
1. for the DNA extraction method of balsam pear purity of hybrid Molecular Identification, it is characterized in that: adopt improved method of CTAB to extract not homophyletic cross-fertilize seed balsam pear tender leaf genome DNA, but without the need to putting into mortar liquid nitrogen grinding, put into-80 DEG C of more than refrigerator 3h, rear pestle grinding about the 5s that mills taken out with cleaning, add rapidly 2 × CTAB Extraction buffer, then chloroform/primary isoamyl alcohol suspends, Virahol and 70% washing with alcohol precipitation, finally obtain a large amount of not homophyletic detected sample balsam pear genomic dna.
2. the DNA extraction method for balsam pear purity of hybrid Molecular Identification according to claim 1, is characterized in that: described method specifically comprises following:
(1) in the different centrifuge tube of 1.5 ml, putting into each 0.1-0.3g of different balsam pear tender leaf sample to be detected respectively, the centrifuge tube that balsam pear tender leaf is housed being placed on flat board, without the need to directly putting into-80 DEG C of freezing 3-5h of refrigerator after lid pipe;
(2) in refrigerator, take out the centrifuge tube that balsam pear tender leaf is housed successively, grinding 5-7 second fast with cleaning the pestle of milling after drying, adding 2 × CTAB Extraction buffer that 500 μ l are preheated to 60 DEG C, fully mixing;
(3) after all samples is milled and added 2 × CTAB Extraction buffer, put into 65 DEG C of water-bath 12-25 min together, period puts upside down mixing 1 time, takes out after water-bath, adds isopyknic chloroform isoamyl alcohol mixed solution in often managing, centrifugal 10 min of 12000r/min;
(4) supernatant liquor 300 μ l is got to another centrifuge tube, add the pH5.2 of equal-volume Virahol and 1/10 volume, 3 mol/L NaAc, place 10 min, centrifugal 5 min of 12000 r/min under 4 DEG C of conditions, white precipitate DNA concentration be 70% washing with alcohol once after, air-dry in super clean bench;
(5) target DNA after drying up is dissolved in 50 μ lTE solution, adds 10 g/L, 0.5 μ LRNase, and in 37 DEG C of incubation 15 min; Namely save backup for Molecular Identification or in-2O DEG C.
3. the DNA extraction method for balsam pear purity of hybrid Molecular Identification according to claim 1, is characterized in that: in step (3), in chloroform isoamyl alcohol mixed solution, each volume components ratio is 24:1.
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Cited By (3)
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| CN105018467A (en) * | 2015-07-17 | 2015-11-04 | 福建农林大学 | Anoectochilus roxburghii DNA extracting method suitable for RAPD analysis |
| CN106834276A (en) * | 2017-02-27 | 2017-06-13 | 广西壮族自治区农业科学院蔬菜研究所 | A kind of balsam pear single seed DNA rapid extracting methods |
| CN111235301A (en) * | 2020-03-23 | 2020-06-05 | 北京市农林科学院 | Method for identifying authenticity of bitter gourd varieties and special SSR primer combination thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105018467A (en) * | 2015-07-17 | 2015-11-04 | 福建农林大学 | Anoectochilus roxburghii DNA extracting method suitable for RAPD analysis |
| CN106834276A (en) * | 2017-02-27 | 2017-06-13 | 广西壮族自治区农业科学院蔬菜研究所 | A kind of balsam pear single seed DNA rapid extracting methods |
| CN111235301A (en) * | 2020-03-23 | 2020-06-05 | 北京市农林科学院 | Method for identifying authenticity of bitter gourd varieties and special SSR primer combination thereof |
| CN111235301B (en) * | 2020-03-23 | 2021-04-27 | 北京市农林科学院 | Method for identifying authenticity of bitter gourd varieties and special SSR primer combination thereof |
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Application publication date: 20150225 |