CN101696408B - Method for extracting DNA from single eggs of copepods - Google Patents
Method for extracting DNA from single eggs of copepods Download PDFInfo
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- CN101696408B CN101696408B CN2009101127009A CN200910112700A CN101696408B CN 101696408 B CN101696408 B CN 101696408B CN 2009101127009 A CN2009101127009 A CN 2009101127009A CN 200910112700 A CN200910112700 A CN 200910112700A CN 101696408 B CN101696408 B CN 101696408B
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Abstract
The invention discloses a method for extracting DNA from single eggs of copepods, relating to a DNA extraction technology. The single eggs have different egg diameters and are generated by different egg producing ways. The method can ensure the extraction efficiency of DNA, and the extracted DNA can be directly applied to downstream PCR experiments. The method comprises the following steps of placing the single eggs of copepods in a 1*TE buffer solution, adding protease K until the final concentration of the protease K reaches 2.5-5.5mg/mLto obtain a mixed extraction solution; carrying out a thermal incubation program on the mixed extraction solution obtained to obtain a crude extract of DNA of the single eggs; and depositing the crude extract of DNA of the single eggs with a nucleic acid coprecipitator to obtain DNA precipitate. The precipitate can be dissolved in sterilized water and stored in a refrigerator for later use.
Description
Technical field
The present invention relates to a kind of DNA extraction technology,, comprise all practical and rapidly effective a kind of novel DNA extraction method in different egg mode, different physiological status and different ovum footpath especially to the single ovum of copepods.
Background technology
At present, the method of extracting at the single ovum of zooplankton mainly contains two kinds in the world, a kind of is (Montero-Pau et al.Application of an inexpensive and high-throughput genomic DNA extractionmethod for the molecular ecology of zooplanktonic diapausing eggs.Limnology and Oceanography:Methods such as Montero-Pau, 2008,6:218-222) the HotSHOT method of Ti Chuing, what this method was primarily aimed at is the extraction of the single ovum genomic dna of zooplankton diapause ovum, and its operation steps mainly comprises following step: add a certain amount of lysis buffer; By physical means and instrument the diapause ovum is smashed up; Incubating with cold process of hitting by a heat at last is released genomic dna.Another kind method is (Walsh et al.Chelex-100 as a medium for simple extraction of DNA forPCR-based typing from forensic material.BioTechniques such as Walsh, 1991,10:506-513) the Chelex-100 method of Ti Chuing, the principle of this method is simple, similar with first method, different is at first to add lysate in the process that this method is extracted, add the Chelex resin of producing by U.S. Bole (Bio-Rad) company subsequently, by physical means ovum is carried out fragmentation, obtained genomic dna the most afterwards by a heat process of incubating then.Certainly, along with continuous progress in science and technology, various commercial reagents boxes arise at the historic moment at small tissue and genes of individuals group DNA extraction, and these test kits include that U.S. Promega company produces
Genomic DNA purification kit, QiagenDNeasy tissue extraction kit that German Qiagen company produces or the like.The principle that the principle of the test kit of the above-mentioned various commerce of mentioning is extracted single ovum genomic dna with two kinds of methods that generally adopt in the world at present is similar, and just all the absorption by film and wash-out are finished collection and purifying to DNA to the commercial reagents box.What deserves to be mentioned is that all there are many problems in present two kinds of generally using at the method for the single ovum DNA extraction of zooplankton and the commercial reagents box that emerged in an endless stream afterwards, conclude get up to mainly contain following some: 1, two kinds of methods of popular all come the structure of ovum is destroyed by the means and the instrument of physics at present, this operation will make the DNA amount of extracting than lacking in theory, these are by the artificial loss that brought of operation, and this loss is huge concerning the not high single ovum of dna content own; 2, in this method of Chelex-100, the resin that itself adds has increased the chance of polluting virtually, and practice has simultaneously proved that the adding of resin has very big restraining effect to downstream PCR experiments; 3, the commercial reagents box mainly process such as absorption by film and wash-out finish collection and the purifying of DNA, elution efficiency directly had influence on the concentration of DNA when the DNA quality was improved, this also will directly have influence on PCR experiment in downstream.
Above-mentioned be external progress in this respect, so far, do not see the report of domestic extraction work to the single ovum genomic dna of copepods.
Summary of the invention
The objective of the invention is to the deficiency when overcoming prior art, the DNA extraction method of the single ovum of a kind of copepods is provided the single ovum extracting genome DNA of single zooplankton.Comprise the single ovum that different ovum footpath and different egg mode are produced.This method not only can guarantee as far as possible that the DNA that the efficient of the extraction of DNA is extracted simultaneously can be applied directly to downstream PCR experiments.
The present invention includes following steps:
1) the single ovum of copepods is placed 1 * TE damping fluid, add Proteinase K, make the final concentration of Proteinase K reach 2.5～5.5mg/mL, get mixed extract;
2) mixed extract that obtains is incubated program by a heat, get the crude extract of single ovum DNA;
3) crude extract to single ovum DNA is precipitated by coprecipitated dose of nucleic acid (DNA Mate), gets the DNA precipitation.Throw out can be dissolved in the aqua sterilisa, be stored in the refrigerator standby.
In step 1), the pH of described 1 * TE damping fluid is preferably 7.5～8.5, the purpose that adds Proteinase K mainly is that the external structure that is used for destroying ovum by Proteinase K makes that inner DNA can be discharged smoothly, the loss of also having avoided physical damage to bring to DNA simultaneously.
In step 2) in, the specific procedure that described heat is incubated program is as follows: place 25～120min, place 5min then under 95 ℃ of temperature for 37～60 ℃.
In step 3), coprecipitated dose of the nucleic acid (DNA Mate) that described nucleic acid coprecipitated dose (DNA Mate) can adopt the precious biological company limited in Dalian (Takara) to produce.
Described sedimentary concrete steps are as follows: add sodium acetate soln and coprecipitated dose of nucleic acid (DNA Mate) in the crude extract of single ovum DNA, continue to add the dehydrated alcohol of-20 ℃ of precoolings, abandon supernatant after the centrifugal 10～30min of 10000～14000rpm, clean and drying with ethanol, promptly get the DNA precipitation.The add-on that described sodium acetate soln and nucleic acid are coprecipitated dose can be by volume single ovum DNA crude extract 10%.The add-on of described dehydrated alcohol can be long-pending 2.5 times of coprecipitated dose three sub-population of crude extract, sodium acetate soln and nucleic acid of single ovum DNA by volume.
The present invention at first utilizes Proteinase K under 55 ℃ of 30min and 95 ℃ of such hot compress programs of 5min single ovum external structure to be carried out cracking, make DNA be able to abundant release, by DNA Mate dna solution is concentrated with precipitation then and can be effectively directly and the single ovum that produced of different egg mode extract genomic dna from the different ovum of copepods.
What nucleic acid coprecipitated dose (DNA Mate) was proved by putting into practice can have very strong precipitation and spissated effect to minim DNA.In the present invention, in the whole process of extracting, reduce as far as possible and use centrifuge tube, can reduce the loss of DNA in the leaching process on the one hand, also can significantly reduce the chance of the pollution of foreign DNA simultaneously.Adopted coprecipitated dose of nucleic acid that DNA is carried out last precipitation among the present invention and concentrate, the use that experimental results show that coprecipitated dose of nucleic acid has improved DNA concentration greatly and the DNA that extracted can directly be applied to the PCR experiment in downstream.
Description of drawings
Fig. 1 is the PCR result of 3 kinds of single ovum genomic dnas of copepods.In Fig. 1, M is GenRuler
TM100bpruler; 1～2 is respectively the PCR result of the single ovum genomic dna of Calanus sinicus and the result of parallel group of PCR; 3～4 are respectively the PCR result of the single ovum genomic dna of thin tail chest thorn water flea and the result of parallel group of PCR; 5～6 are respectively the PCR result of the single ovum genomic dna of mother-in-law Luo Yi Cyclop and the result of parallel group of PCR; 7 are the PCR result of blank sample (replacing single ovum to experimentize with ultrapure water).
Embodiment
The single ovum of copepods is placed 1 * TE damping fluid (pH 8) of 5～10 μ L; Add Proteinase K, make the final concentration of Proteinase K reach 4mg/mL (purpose that adds Proteinase K mainly is that the external structure that is used for destroying ovum by Proteinase K makes that inner DNA can be discharged smoothly, the loss of also having avoided physical damage to bring to DNA simultaneously); Just can obtain mixed extract after adding Proteinase K, incubate program with the extracting solution that mixes that is about to obtain by a heat, specific procedure is as follows: place 30min, place 5min then under 95 ℃ of temperature for 55 ℃.Just can obtain the crude extract of single ovum DNA after said procedure finishes, next precipitate by the nucleic acid of producing by the precious biological company limited in Dalian (Takara) coprecipitated dose (DNA Mate).Sedimentary concrete steps are as follows: the sodium acetate soln and the 4 μ LDNA Mate that add the 3M of DNA crude extract 1/10th volumes; The dehydrated alcohol of-20 ℃ of precoolings of 2.5 times of volumes of the above-mentioned overall solution volume of continuation adding is abandoned supernatant after the centrifugal 20min of 12000rpm; With volume ratio is that 70% ethanol cleans and dry; Obtain the DNA precipitation after dry, at last this DNA precipitation is dissolved in be stored in-20 ℃ of refrigerators in the aqua sterilisa of 5～20 μ L standby.What nucleic acid coprecipitated dose (DNA Mate) was proved by putting into practice can have very strong precipitation and spissated effect to minim DNA.In the present invention, in the whole process of extracting, reduce as far as possible and use centrifuge tube, can reduce the loss of DNA in the leaching process on the one hand, also can significantly reduce the chance of the pollution of foreign DNA simultaneously.Adopted coprecipitated dose of nucleic acid that DNA is carried out last precipitation among the present invention and concentrate, the use that experimental results show that coprecipitated dose of nucleic acid has improved DNA concentration greatly and the DNA that extracted can directly be applied to the PCR experiment in downstream.
The present invention is to 3 kinds of different types of copepods in utilization, is respectively: thin tail chest thorn water flea (Thompson﹠amp; Scott, 1903), Calanus sinicus (Brodsky, 1962) and mother-in-law Luo Yi Cyclop (Lindberg, 1954) have carried out the extraction of single ovum genomic dna, wherein thin tail chest thorn water flea and Calanus sinicus belong to the copepods of the type of freely laying eggs, and mother-in-law Luo Yi Cyclop then belongs to band egg capsule type.Every kind of copepods be provided with two groups parallel, the single ovum genomic dna that will extract at last is directly used in the PCR experiment, primer CopF2 and CopR1 can amplify copepods 28S rDNA specific fragment (～300bp).PCR the results are shown in accompanying drawing, and wherein M is GenRuler
TM100bp ruler; 1～2 is respectively the PCR result of the single ovum genomic dna of Calanus sinicus and the result of parallel group of PCR; 3～4 are respectively the PCR result of the single ovum genomic dna of thin tail chest thorn water flea and the result of parallel group of PCR; 5～6 are respectively the PCR result of the single ovum genomic dna of mother-in-law Luo Yi Cyclop and the result of parallel group of PCR; 7 are the PCR result of blank sample (replacing single ovum to experimentize with ultrapure water).The result shows the extraction of utilization the present invention to the single ovum genomic dna of different sorts copepods, comprises the genomic extraction of single ovum to mode with the different ovum footpath size of different egg.Resulting single ovum genomic dna can directly apply to the PCR reaction in downstream, and the situation that no foreign DNA pollutes in experiment appearance, and entire operation is simple and rapidly efficient.
Similar to Example 1, the single ovum of copepods is placed 1 * TE damping fluid (pH 7.5) of 5～10 μ L; Add Proteinase K, make the final concentration of Proteinase K reach 2.5mg/mL (purpose that adds Proteinase K mainly is that the external structure that is used for destroying ovum by Proteinase K makes that inner DNA can be discharged smoothly, the loss of also having avoided physical damage to bring to DNA simultaneously); Just can obtain mixed extract after adding Proteinase K, incubate program with the extracting solution that mixes that is about to obtain by a heat, specific procedure is as follows: place 25min, place 5min then under 95 ℃ of temperature for 60 ℃.Just can obtain the crude extract of single ovum DNA after said procedure finishes, next precipitate by the nucleic acid of producing by the precious biological company limited in Dalian (Takara) coprecipitated dose (DNA Mate).Sedimentary concrete steps are as follows: the sodium acetate soln and the 4 μ L DNA Mate that add the 3M of DNA crude extract 1/10th volumes; The dehydrated alcohol of-20 ℃ of precoolings of 2.5 times of volumes of the above-mentioned overall solution volume of continuation adding is abandoned supernatant after the centrifugal 30min of 10000rpm; With volume ratio is that 70% ethanol cleans and dry; Obtain the DNA precipitation after dry, at last this DNA precipitation is dissolved in be stored in-20 ℃ of refrigerators in the aqua sterilisa of 5～20 μ L standby.
Similar to Example 1, the single ovum of copepods is placed 1 * TE damping fluid (pH 8.5) of 5～10 μ L; Add Proteinase K, make the final concentration of Proteinase K reach 5.5mg/mL (purpose that adds Proteinase K mainly is that the external structure that is used for destroying ovum by Proteinase K makes that inner DNA can be discharged smoothly, the loss of also having avoided physical damage to bring to DNA simultaneously); Just can obtain mixed extract after adding Proteinase K, incubate program with the extracting solution that mixes that is about to obtain by a heat, specific procedure is as follows: place 120min, place 5min then under 95 ℃ of temperature for 37 ℃.Just can obtain the crude extract of single ovum DNA after said procedure finishes, next precipitate by the nucleic acid of producing by the precious biological company limited in Dalian (Takara) coprecipitated dose (DNA Mate).Sedimentary concrete steps are as follows: the sodium acetate soln and the 4 μ L DNA Mate that add the 3M of DNA crude extract 1/10th volumes; The dehydrated alcohol of-20 ℃ of precoolings of 2.5 times of volumes of the above-mentioned overall solution volume of continuation adding is abandoned supernatant after the centrifugal 10min of 14000rpm; With volume ratio is that 70% ethanol cleans and dry; Obtain the DNA precipitation after dry, at last this DNA precipitation is dissolved in be stored in-20 ℃ of refrigerators in the aqua sterilisa of 5～20 μ L standby.
Claims (4)
1. the DNA extraction method of the single ovum of copepods is characterized in that may further comprise the steps:
1) the single ovum of copepods is placed 1 * TE damping fluid, add Proteinase K, make the final concentration of Proteinase K reach 2.5～5.5mg/mL, get mixed extract;
2) mixed extract that obtains is incubated program by a heat, get the crude extract of single ovum DNA, the specific procedure that described heat is incubated program is as follows: place 25～120min, place 5min then under 95 ℃ of temperature for 37～60 ℃;
3) crude extract to single ovum DNA is precipitated for coprecipitated dose by nucleic acid, gets the DNA precipitation.
2. the DNA extraction method of the single ovum of copepods as claimed in claim 1 is characterized in that in step 1), and the pH of described 1 * TE damping fluid is 7.5～8.5.
3. the DNA extraction method of the single ovum of copepods as claimed in claim 1, it is characterized in that in step 3), described sedimentary concrete steps are as follows: add coprecipitated dose of sodium acetate soln and nucleic acid in the crude extract of single ovum DNA, continue to add the dehydrated alcohol of-20 ℃ of precoolings, abandon supernatant after the centrifugal 10～30min of 10000～14000rpm, clean and drying with ethanol, promptly get the DNA precipitation.
4. the DNA extraction method of the single ovum of copepods as claimed in claim 3, the add-on that it is characterized in that described dehydrated alcohol are 2.5 times that coprecipitated dose three sub-population of crude extract, sodium acetate soln and nucleic acid of single ovum DNA amasss by volume.
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| CN102250882B (en) * | 2011-06-27 | 2012-12-05 | 中国科学院南海海洋研究所 | Method for extracting DNA (Deoxyribonucleic Acid) of total genome from zooplankter and intestinal inclusions thereof |
| CN103937782A (en) * | 2014-04-29 | 2014-07-23 | 天津农学院 | Fast extraction method of genomic DNA of single rotifer |
| CN117126843B (en) * | 2023-09-18 | 2024-05-14 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | DNA extraction method for small zooplankton single body |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100710418B1 (en) * | 2006-02-09 | 2007-04-24 | 단국대학교 산학협력단 | Dna extraction strip and dna extraction method comprising the same |
| CN101423542A (en) * | 2008-12-12 | 2009-05-06 | 广州中医药大学 | Extraction method of cubilose whole genome DNA |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100710418B1 (en) * | 2006-02-09 | 2007-04-24 | 단국대학교 산학협력단 | Dna extraction strip and dna extraction method comprising the same |
| CN101423542A (en) * | 2008-12-12 | 2009-05-06 | 广州中医药大学 | Extraction method of cubilose whole genome DNA |
Non-Patent Citations (2)
| Title |
|---|
| Montero-Pau et al..Application of an inexpensive and high-throughput genomic DNA extraction method for the molecular ecology of zooplanktonic diapausing eggs.《Limnology and Oceanography：Methods》.2008,第6卷218-222. * |
| 王桂忠等.海洋浮游桡足类滞育及其生理生态学研究进展.《厦门大学学报(自然科学版)》.2006,第45卷(第2期),46-53. * |
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