CN101748174A - DNA rapid extraction method for single-seed of sorghum halepense and similar species - Google Patents
DNA rapid extraction method for single-seed of sorghum halepense and similar species Download PDFInfo
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- CN101748174A CN101748174A CN200810072359A CN200810072359A CN101748174A CN 101748174 A CN101748174 A CN 101748174A CN 200810072359 A CN200810072359 A CN 200810072359A CN 200810072359 A CN200810072359 A CN 200810072359A CN 101748174 A CN101748174 A CN 101748174A
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Abstract
The invention discloses a method for directly and rapidly extracting DNA from single-seed of sorghum halepense and similar species. DNA is directly extracted from the single-seed of the sorghum halepense and the similar species, and the obtained quality is compared with the DNA extracted from leaves of young seedling, from the point of view of the calculated purity through OD value or the result of electrophoresis verification, no significant difference exists, and the DNA meets the condition serving as PCR amplified template through testing. The invention simplifies the steps for extracting DNA from the single-seed of sorghum halepense and similar species, and shortens the time for extracting the DNA, and adopts the method for directly extracting DNA from the single-seed, and omits the germination process, and greatly saves time, therefore, the invention has important application value in the identification of species authenticity through using DNA fingerprint.
Description
Technical field
The present invention relates to be used for the method that the crop varieties seed is identified, relate in particular to a kind of from false Chinese sorghum of simple grain and allied species seed thereof the direct method of rapid extraction minim DNA, belong to the technical field that seed authenticity is identified.
Background technology
False Chinese sorghum is one of the world's ten big malignant weeds, and China has listed it in the harmful organism list.It is quite similar that Sorghum such as seed characteristics of false Chinese sorghum and plan Chinese sorghum, arabian cron belong to other kinds, the false sorghum seeds of port intercepting and capturing is because rachilla in addition, characteristic portion such as grain husk shell etc. easily be extruded and fracture, breakage, giving seed and be accredited as main port quarantine work and bring bigger difficulty, is a kind of simple and reliable authentication method with PCR method Rapid identification vacation Chinese sorghum and allied species thereof therefore.But can provide the sample size of Molecular Identification only to be 1-2 grain seed in the port quarantine work usually, traditional DNA extraction method and existing test kit did not all propose the extraction scheme to micro-seed sample like this.
Existing DNA extraction method has SDS method, CTAB method, test kit method.Guo Qiongxia etc. had compared four kinds of extracting method (SDS method, CTAB method, moller method, TAKARA test kit) (Guo Qiongxia etc. of the DNA of false Chinese sorghum and seven allied specieses, the DNA trace extracting method that Sorhum belongs to 7 allied specieses compares, Plant Quarantine [J], 2005,19 (2): 65-68), obtain conclusion: to the extraction of limited quarantine injurious weed sample (0.05g-0.1g) DNA, adopt the extraction yield of Moller method the highest, be respectively 2.11 times of SDS method, 1.17 times of CTAB method, 5.8 times of takara test kit.
DNA of plants extraction test kit on the market exceeds with the weight of 0.05g mostly at present, and the average single seed amount of the seed of false Chinese sorghum is 0.005g, and promptly existing test kit can not guarantee to satisfy the more demand of micro-example DNA of extracting.
The objective of the invention is to avoid the sample size long deficiency still bigger and consuming time of the traditional method of DNA extraction and available reagent box and provide a kind of and can from single seed, extract the DNA extraction method that is enough to satisfy later stage Molecular Identification requirement.Present method can extract the DNA of 30ul220ng/ul from the false Chinese sorghum mature seed of simple grain.And the DNA demand of a PCR is about 50ng, and promptly present method is extracted to such an extent that single seed DNA amount can satisfy the demand of doing 130 PCR detections at least.
Guo Qiong rosy clouds study group gets method to many seed DNA extraction of Sorghum 7 allied specieses of genus and reports in " the DNA trace extracting method that Sorghum belongs to 7 allied specieses compares " literary composition.The present invention continues to explore Sorghum to belong to 7 allied species single seed DNA extraction rates and dna purity and extraction time gained result after above-mentioned research.The moller method of report in 2005 such as Guo Qiongxia " the DNA trace extracting method that Sorghum belongs to 7 allied specieses relatively " is listed: sample thief 0.05g herein, behind liquid nitrogen flash freezer, be ground to powder, add 500 μ lTES[100mM Tris, 10mM EDTA, 2%SDS], add 50-100 μ g Proteinase K, 55-60 ℃ of incubation 0.5-1h, during shake mixing gently; Regulate NaCl concentration to 1.4mol/L, add 1/10 volume 10%CTAB, 65 ℃ of 10min; Add 1 volume chloroform: primary isoamyl alcohol (24: 1), the jog mixing, in 0 ℃ of 30min, 4 ℃ of centrifugal 10min of 12000rpm afterwards; Get supernatant, add 225 μ l 5molNH4AC, mixing is placed on ice more than the 30min 4 ℃ of centrifugal 10min of 12000rpm; Get supernatant, add 3ul Rnase, in 37 ℃ of 15min, 4 ℃ of 12000 centrifugal 10min; Get supernatant, add 0.55 volume isopropanol precipitating DNA, if the centrifugal 5min of 12000rpm immediately the appearance of no DNA group, puts sample immediately in 15-30min on ice; Abandon supernatant, 70% ethanol is washed precipitation 2 times, is dissolved in after the drying among the 50 μ lTE.
Summary of the invention
The purpose of present method is to make every effort to extract the highly purified DNA of high density in the shortest time from false Chinese sorghum and allied species single seed thereof.
The method of false Chinese sorghum and the direct rapid extraction DNA of allied species single seed thereof comprises the steps:
(1) gets false Chinese sorghum or its allied species simple grain mature seed, behind liquid nitrogen flash freezer, be ground to powder;
(2) in aforementioned powder, successively add TES and Proteinase K, 55-60 ℃ of incubation;
(3) adding concentration is 1.4mol/LNaCl, then, adds 1/10 volume 10%CTAB, incubation;
(4) past step (3) adds the chloroform of 1 volume: primary isoamyl alcohol, the volume ratio of chloroform and primary isoamyl alcohol are 24: 1, mixing, and ice is fully centrifugal after educating;
(5) get aforementioned centrifugal supernatant liquor afterwards, add an amount of NH
4AC, ice is fully centrifugal after educating;
(6) get centrifugal supernatant liquor afterwards in aforementioned (5), add 0.55 volume isopropanol precipitating DNA, fully centrifugal immediately, if no DNA group occurs, immediately centrifuge tube is placed on ice;
(7) abandon the supernatant liquor of step (6), ethanol is washed precipitation, is dissolved among the TE after the drying.
In the preceding method, it is 8.0 Tris that described TES extracting solution contains 100mM pH, and 10mM pH is that 8.0 EDTA and mass concentration are 2% SDS.
The present invention need not the process through germinateing, and only needs single seed to grind extraction, and sample size is trace extremely, can extract the DNA of 30ul 220ng/ul from the false sorghum seeds kind of 0.005g.Existing commercially available vegetable material minim DNA extracts the sample size of test kit in specification sheets and all is not less than 0.05g.The omnidistance time that this law is extracted DNA is about 200min.Can satisfy the requirement of port quarantine rapid detection fully, simplify process greatly, save the time, be a kind of very convenient, economy, effective means.In false jowar and the research of allied species seed authenticity Rapid identification thereof, the present invention has great application value.
Description of drawings
Fig. 1 is the electrophoretic effect of plant leaf rbcL-PCR.
Fig. 2 is the electrophoretic effect of single seed rbcL-PCR.
Among Fig. 1, Fig. 2: numeral 1 to 10 is represented false jowar, black Chinese sorghum, silk gram Chinese sorghum respectively, is intended Chinese sorghum, No. 1, bright good fortune, arabian cron, Gao Dancao, two look Chinese sorghums, light jowar, clear water contrast.The rbcL primer sequence is:
5’-ATGTCACCACAAACAGA-3’，
5’-GATTGGGCCGAGTTTAATT-3’。
Embodiment
The method of false Chinese sorghum and the direct rapid extraction DNA of allied species single seed thereof comprises the steps:
(1) gets 1 false Chinese sorghum or its allied species simple grain mature seed, behind liquid nitrogen flash freezer, be ground to powder;
(2) add 500 μ lTES (it is 8.0 Tris that the TES extracting solution contains 100mM pH, and 10mM pH is that 8.0 EDTA and mass concentration are 2% SDS), add 50-100 μ g Proteinase K, 55-60 ℃ of incubation 1h, during shake mixing gently;
(3) regulate NaCl concentration to 1.4mol/L, add 1/10 volume 10%CTAB, 65 ℃ of 10min;
(4) add 1 volume chloroform: primary isoamyl alcohol (24: 1), the jog mixing, in 0 ℃ of 30min, 4 ℃ of centrifugal 10min of 12000rpm afterwards;
(5) get supernatant, add 225 μ l 5mol NH
4AC, mixing is placed on 1h on ice, 4 ℃ of centrifugal 10min of 12000rpm;
(6) get supernatant, add 0.55 volume isopropanol precipitating DNA, if the centrifugal 5min of 12000rpm immediately the appearance of no DNA group, puts sample immediately in 15-30min on ice;
(7) abandon supernatant, 70% ethanol is washed precipitation 2 times, is dissolved in after the drying among the 30 μ lTE.
Reported method difference in 2005 such as the present invention and Guo Qiongxia: present method is clear and definite adds the time of water-bath behind the Proteinase K and adds NH
4The time of ice bath behind the Ac, the water-bath and the ice bath time of optimum extraction effect have been determined; And according to the actual needs of Molecular Identification, the step of Rnase is added in deletion.
Single seed to 9 sorghums (Sorghum) plants such as false jowar, black Chinese sorghum, silk gram Chinese sorghum, plan Chinese sorghum, No. 1, bright good fortune, arabian cron, Gao Dancao, two look Chinese sorghums, light jowar has carried out DNA extraction.Step is as follows:
(1) gets 1 false Chinese sorghum or its allied species simple grain mature seed, behind liquid nitrogen flash freezer, be ground to powder;
(2) add 500 μ lTES[100mM Tris, 10mM EDTA, 2%SDS], add 50-100 μ g Proteinase K, 55-60 ℃ of incubation 1h, during shake mixing gently;
(3) regulate NaCl concentration to 1.4mol/L, add 1/10 volume 10%CTAB, 65 ℃ of 10min;
(4) add 1 volume chloroform: primary isoamyl alcohol (24: 1), the jog mixing, in 0 ℃ of 30min, 4 ℃ of centrifugal 10min of 12000rpm afterwards;
(5) get supernatant, add 225 μ l 5mol NH
4AC, mixing is placed on 1h on ice, 4 ℃ of centrifugal 10min of 12000rpm;
(6) get supernatant, add 0.55 volume isopropanol precipitating DNA, if the centrifugal 5min of 12000rpm immediately the appearance of no DNA group, puts sample immediately in 15-30min on ice;
(7) abandon supernatant, 70% ethanol is washed precipitation 2 times, is dissolved in after the drying among the 30 μ lTE.
In order relatively to propose the effect of DNA from blade, the bright leaf to 9 species among the embodiment one carries out DNA extraction simultaneously, and each species is got the bright leaf of 0.05g, and step is the same.Under nucleic acid egg hundred analysers, measured their OD260, OD280, OD260/280 and concentration (ng/ul), and the DNA that extracts has been carried out the rbcL-PCR agarose gel electrophoresis detect.With the OD260/280 value is the purity that index is weighed DNA, and it is purer relatively that this value equals 1.8 expression DNA; Less than among the 1.8 expression DNA protein contamination being arranged; Greater than having RNA to pollute among 1.8 expression DNA.From measurement result (table 1 of the present invention, table 2), the DNA OD260/280 value of extracting from false jowar and 8 allied species single seeds and seedling leaves is all very near 1.8, illustrates that the DNA purity that the present invention extracts false jowar of simple grain and allied species seed thereof meets the requirements.
Table 1 extracts the OD value of DNA from false jowar and allied species seedling leaves thereof
| Species | ??OD 260/280 | ??OD 260/230 | Concentration (ng/ul) |
| False jowar | ??1.726 | ??1.026 | ??369 |
| Black Chinese sorghum | ??1.664 | ??0.441 | ??134 |
| Silk gram Chinese sorghum | ??1.754 | ??1.239 | ??167 |
| Intend Chinese sorghum | ??1.831 | ??1.232 | ??239 |
| Species | ??OD 260/280 | ??OD 260/230 | Concentration (ng/ul) |
| No. 1, bright good fortune | ??1.820 | ??0.884 | ??226 |
| Arabian cron | ??1.703 | ??0.875 | ??271 |
| Gao Dancao | ??1.735 | ??0.898 | ??275 |
| Two look Chinese sorghums | ??1.802 | ??1.165 | ??343 |
| The light Chinese sorghum | ??1.903 | ??1.974 | ??140 |
Table 2 extracts the OD value of DNA from false jowar and allied species single seed thereof
| Species | ??OD 260/280 | ??OD 260/230 | Concentration (ng/ul) |
| False jowar | ??1.82 | ??1.28 | ??220 |
| Black Chinese sorghum | ??1.82 | ??1.10 | ??232 |
| Silk gram Chinese sorghum | ??1.66 | ??1.12 | ??180 |
| Intend Chinese sorghum | ??1.910 | ??1.243 | ??210 |
| No. 1, bright good fortune | ??1.973 | ??1.269 | ??187 |
| Arabian cron | ??1.636 | ??1.331 | ??225 |
| Gao Dancao | ??1.935 | ??1.626 | ??338 |
| Two look Chinese sorghums | ??1.812 | ??1.165 | ??380 |
| The light Chinese sorghum | ??1.974 | ??1.303 | ??190 |
9 the Sorghum platymiscium seeds extracting and the DNA of blade are diluted to 50ng/ul, carry out the rbcL-PCR agarose gel electrophoresis and detect, present clear, consistent segment at the 400bp place.No matter the measurement result of bind nucleic acid protein analyzer and electrophoretogram are the DNA of seed extraction or the DNA that plant leaf extracts, and all meet the test requirements document as the pcr amplification template.
Claims (2)
1. the method for false Chinese sorghum and the direct rapid extraction DNA of allied species single seed thereof comprises the steps:
(1) gets false Chinese sorghum or its allied species simple grain mature seed, behind liquid nitrogen flash freezer, be ground to powder;
(2) in aforementioned powder, successively add TES and Proteinase K, 55-60 ℃ of incubation;
(3) adding concentration is 1.4mol/LNaCl, then, adds 1/10 volume 10%CTAB, incubation;
(4) past step (3) adds the chloroform of 1 volume: primary isoamyl alcohol, the volume ratio of chloroform and primary isoamyl alcohol are 24: 1, mixing, and ice is fully centrifugal after educating;
(5) get aforementioned centrifugal supernatant liquor afterwards, add an amount of NH4AC, ice is fully centrifugal after educating;
(6) get centrifugal supernatant liquor afterwards in aforementioned (5), add 0.55 volume isopropanol precipitating DNA, fully centrifugal immediately, if no DNA group occurs, immediately centrifuge tube is placed on ice;
(7) abandon the supernatant liquor of step (6), ethanol is washed precipitation, is dissolved among the TE after the drying.
2. as the method for claims 1 described false Chinese sorghum and the direct rapid extraction DNA of allied species single seed thereof, it is characterized in that: it is 8.0 Tris that described TES extracting solution contains 100mM pH, and 10mM pH is that 8.0 EDTA and mass concentration are 2%SDS.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592077A (en) * | 2019-10-28 | 2019-12-20 | 云南师范大学 | DNA extracting solution of pedunculate herpetospermum seeds, extraction kit, application of DNA extracting solution and extraction method of DNA extracting solution |
| CN117757984A (en) * | 2024-02-22 | 2024-03-26 | 中国热带农业科学院三亚研究院 | DNA probe, kit and method for identifying sorghum halepense |
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- 2008-12-12 CN CN200810072359A patent/CN101748174A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592077A (en) * | 2019-10-28 | 2019-12-20 | 云南师范大学 | DNA extracting solution of pedunculate herpetospermum seeds, extraction kit, application of DNA extracting solution and extraction method of DNA extracting solution |
| CN117757984A (en) * | 2024-02-22 | 2024-03-26 | 中国热带农业科学院三亚研究院 | DNA probe, kit and method for identifying sorghum halepense |
| CN117757984B (en) * | 2024-02-22 | 2024-05-14 | 中国热带农业科学院三亚研究院 | DNA probe, kit and method for identifying sorghum halepense |
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