CN101671731A - Simple, rapid and efficient agarose gel electrophoresis method for detecting plant molecular marker - Google Patents
Simple, rapid and efficient agarose gel electrophoresis method for detecting plant molecular marker Download PDFInfo
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- CN101671731A CN101671731A CN200910160394A CN200910160394A CN101671731A CN 101671731 A CN101671731 A CN 101671731A CN 200910160394 A CN200910160394 A CN 200910160394A CN 200910160394 A CN200910160394 A CN 200910160394A CN 101671731 A CN101671731 A CN 101671731A
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Abstract
The invention provides a simple and rapid agarose gel electrophoresis method suitable for rapid detection of genetic variation of a high-throughput plant SSR marker locus. The method comprises the following steps: performing further treatment to the gel after detection by agarose gel electrophoresis, and performing detection to the next molecular marker PCR product for the next round through the treated gel. The gel can be reused after treatment, one piece of gel can be reused for more than 10 times, an electrophoresis strip can be still ensured clear, the resolution ratio is high, and the repeatability is good. Therefore, the invention realizes the rapid detection to the genetic variation of the high-throughput plant SSR marker locus, simultaneously overcomes the defects existing in the existing agarose gel electrophoresis method with excessive waste of the reagent medicine, harm to the human body from the coloring agent and serious pollution of gel and wastes of electrophoretic buffer to the environment, greatly improves the utilization efficiency of the agarose gel and the electrophoretic buffer and saves the man power as well as the cost.
Description
Technical field
The present invention relates to a kind of agarose gel electrophoresis detection method simply, fast and efficiently, this method is particularly useful for the detection of plant molecular marker.
Background technology
In recent years, the plant molecular marker technical development is rapid, and has brought into play vital role in research fields such as the genetic mapping of Main Agronomic Characters, the assignment of genes gene mapping, gene clone, molecular mark, analysis of genetic diversity, sibship evaluation, the predictions of hybrid vigour mechanism.At present, be widely used in plant molecular marker detection technique system and comprise polyacrylamide gel and agarose gel electrophoresis, the two is all by the migration distance of comparison object molecule marker amplified band on gel, and learns the length polymorphism on certain molecule marker seat between Different Individual.But, conventional polyacrylamide gel electrophoresis from the washing of sheet glass, dry, glue, encapsulating, solidify, electrophoresis, dyeing process color to the end need 4～5 hours, time-consuming when carrying out the high-throughput experiment, effort have a strong impact on whole experiment progress.Shortcomings such as although some polyacrylamide gel electrophoresis methods reports through improvement are arranged, it is many still to exist operation steps, and long or resolving power of time is low.Recently, though abroad have some companies to develop or developing efficient polyacrylamide gel electrophoresis detector,, be difficult to extensively popularizing because plant and instrument is very expensive.
Agarose is a kind of assorted poly-polysaccharide that extracts from marine alga, is the wire superpolymer that is alternately linked to each other and form with α-1,3 and β-1,4 glycosidic link by D type and L type semi-lactosi.This polysaccharide is met cold water and is expanded, and is dissolved in hot water and becomes colloidal sol, can form after the cooling to have inflexible filter opening gel, and to greater than the 200nm, its size is decided by the concentration of agarose to pore diameter range from 50nm, and the big more filter opening of concentration aperture is more little.Agarose gel electrophoresis is as upholder with agar gel, utilize the electrocharge effect of nucleic acid molecule in being higher than the electrophoretic buffer of its iso-electric point and the molecular sieve effect of gel, make the mobility of the nucleic acid molecule that size is different with conformation occur, thereby reach isolating purpose than big-difference.
Sepharose is strong because of its wetting ability, physicochemical property stable and preparation process is very simple (when preparing the gel of different concns, only need take by weighing the agarose powder dissolution of respective numbers in the electrophoretic buffer of certain volume, in boiling water bath or microwave oven, heat and fully melt, be cooled to 50～60 ℃, add ethidium bromide (Ethidium Bromide, EB) staining agent mixing, pour into horizontal rubber moulding, treat can carry out electrophoresis after it fully solidifies) and become one of separation, evaluation and purifying DNA fragment method the easiest and commonly used.Sepharose seldom reclaims and reuses it in laboratory because gel is made easily, normally promptly is dropped after electrophoresis end, ultraviolet gel imaging system record electrophoresis result.This way has caused a large amount of wastes of agarose powder and electrophoretic buffer on the one hand, has increased the cost of analytical test significantly; The EB that is added in the process of preparation gel is a kind of strong carcinogen on the other hand, and long-term contact meeting causes certain injury to human body; At last, when processing contains the gel of EB and electrophoretic buffer, not only bring many troubles, and also can cause great pollution environment to the laboratory.Therefore, development and optimization agarose gel electrophoresis method for detecting are the problems of a worth special concern.
Summary of the invention
What the objective of the invention is to propose in order to solve a difficult problem that exists in the above-mentioned agarose gel electrophoresis a kind ofly has broad applicability and is particularly useful for the agarose gel electrophoresis method for detecting fast and efficiently that the heritable variation of high-throughput plant SSR marker site detects.Described method comprises the step that the gel after detecting through agarose gel electrophoresis is further handled, specific as follows, after last round of detection, the gel that has write down image is placed in the electrophoresis chamber, connecting power supply runs out of the DNA sample outside the glue, gel is washed, to remove interference the next round electrophoresis detection; Dye the glue step then, the gel after having washed is immersed in contains in the electrophoresis buffered soln that final concentration is 0.5 μ g/ml EB 2 hours, described gel promptly can be used for the detection of next round to next molecule marker PCR product through after the above-mentioned processing.
Selectable, for easy to operate, with the gel that write down image with minor operation cutter rectangular about the 25 * 4.5cm that along the direction parallel gel is cut into four equalizations above the point sample hole with the point sample hole, the integrity that note to keep the point sample hole, with its parallel equidistant in being placed on electrophoresis chamber.
Selectable, described agarose gel electrophoresis detects and comprises glue, electrophoresis, detection step, the glue process is as follows, be ready to rubber moulding, specification 25 * the 20cm of gel pallet, the equidistant parallel four row's combs of inserting, take by weighing 7.5g agarose powder to the erlenmeyer flask of 500ml, add in 250ml 0.5 * TBE electrophoresis buffered soln, shake up, in microwave oven, be heated to agarose and be melted into colloidal sol fully, be cooled to below 60 ℃, adding 50 μ l final concentrations again in bottle is the EB of 0.5 μ g/ml, fully mixing, pour colloidal sol into rubber moulding, promptly become gel after solidifying; Electrophoresis process is as follows, carefully extract the comb that is inserted in the gel, gel is put into electrophoresis chamber, add 0.5 * TBE electrophoresis buffered soln to liquid level and cover gel 1-2mm, draw the PCR product that contains sample-loading buffer of 4.5 μ l and carefully add the comb hole with moving the liquid volley of rifle fire, after treating to finish sample on all samples, connect power supply, regulating voltage is to 4-5V/cm, nucleic acid molecule moves on to positive pole from negative pole, when treating that sample-loading buffer is gone to correct position, stop electrophoresis, need 45-60min approximately; Testing process is as follows, and the sepharose that electrophoresis is finished places in the middle of the platform of gel automated imaging system, opens UV-light, can see the DNA band that sends fluorescence, observes and preserve image in computer.
Gel can recycle through after the described processing, and a clotting glue can reuse more than 10 times, can guarantee that still electrophoretic band is clear, resolving power height, good reproducibility.
Agarose gel electrophoresis method for detecting of the present invention is that used sepharose is further run processing such as glue, washing, dyeing, realizes the recycling of gel, thereby has satisfied the rapid detection of high-throughput plant SSR marker site heritable variation.
The agarose gel electrophoresis method for detecting fast and efficiently that the present invention proposes, be wetting ability, physics and chemistry stability of structure and the high resolving power of utilizing sepharose and a kind of new agarose gel electrophoresis method for detecting that forms, this method does not have specificity to plant, need be as long as the plant that is studied has by the molecule marker of electrophoretic separation, all can adopt this method that the idiotype of being studied is made a distinction, so have extensive applicability.
The rapid detection of high-throughput plant SSR marker site of the present invention heritable variation is finished according to following operation steps: it is standby at first to extract plant genome DNA; Develop then and synthesize molecule marker in this plant, utilize these primers that the genomic dna of Different Individual in different groups or the colony is carried out pcr amplification (polymerase chain reaction), (specification of gel pallet is 25 * 20cm to amplified production at 3% the sepharose that contains EB, four row's combs) go up electrophoresis detection, with ultraviolet gel imaging system record electrophoresis result, first run electrophoresis finishes; Electrophoretic gel cuts into gel four equalizations line by line along the direction parallel with the point sample hole above the point sample hole with the minor operation cutter rectangular (25 * 4.5cm will be stopped, note keeping the integrity in point sample hole), be placed in the electrophoresis chamber microscler gel is parallel, connecting power supply continuation electrophoresis washs gel, DNA sample in gel is run out of outside the glue, to remove the interference to PCR product detection next time; At last, the gel that has washed is immersed in contains in the electrophoresis buffered soln that final concentration is 0.5 μ g/ml EB 2 hours, can reuse, carry out the detection of next round PCR product.Because gel can repeatedly reuse, thereby realized the rapid detection of high-throughput plant SSR marker site heritable variation.
Beneficial effect of the present invention:
(1) overcome the excess waste, the staining agent that exist reagent chemicals in the existing agarose gel electrophoresis method for detecting in domestic laboratory the harm of human body and the waste of gel and electrophoretic buffer have been produced the shortcoming of greatly polluting to environment, improve the utilising efficiency of sepharose and electrophoretic buffer greatly, saved manpower and cost.Do not need to prepare again gel when analyzing not isolabeling, a glue can reuse 10 times at least, carries out the detection of 10 above marks, only need about 4 yuan agar Icing Sugar, and conventional sense need spend more than 40 yuan, has saved the cost more than 10 times.
(2) by to the washing of gel with dye glue, avoided the crossed contamination of sample room when repeatedly reusing gel and the reduction of EB concentration to cause image blur unclear.Through the gel that washs and dyeed, electrophoretic band is clear, resolving power height, good reproducibility.
(3) identify fast, comprise that with one of 300 SSR labeled analysis the heritable variation of the plant segregating population of 200 strain systems only needs 1.5 months time.The present invention adopts improved agarose gel electrophoresis method for detecting to simplify the gel making step greatly, significantly improve detection efficiency, be widely used in the SSR Markers for Detection in fields such as plant genetic mapping, the assignment of genes gene mapping and clone, molecular mark, analysis of genetic diversity.
In sum, the sepharose of the present invention after to electrophoresis utilizes after running glue, washing and dyeing once again, and verified by the jowar segregating population being carried out a large amount of SSR molecule markers, significantly reduced the use of agarose powder and electrophoretic buffer, save time, the man power and material of preparation gel, also guaranteed high-quality resolving effect and repeatability simultaneously.Compare and utilize polyacrylamide gel electrophoresis that the plant segregating population is carried out a large amount of Markers for Detection to have bigger time and cost advantage.The foundation of this platform, efficient SSR molecule marker agarose gel electrophoresis detection method in the fields such as relevant plant genetic mapping, molecular mark and germ plasm resource analysis of genetic diversity not only can be provided for the investigator, but also can create a resource-conserving and environmentally friendly laboratory.The present invention has simultaneously expanded the function usefulness and the range of application of agarose gel electrophoresis, has very big commercial value, and businessman can directly prepare and sell the precast gel of different agarose concentrations for meeting the need of market on the basis of this invention.
Description of drawings
Fig. 1: the schema when agarose gel electrophoresis method for detecting of the present invention is used for the plant molecular marker detection;
Fig. 2: show the right agarose gel electrophoresis result of first primer of the first round;
Fig. 3: show that second takes turns second agarose gel electrophoresis result that primer is right;
Fig. 4: show the 3rd right agarose gel electrophoresis result of primer of third round;
Fig. 5: show the 4th right agarose gel electrophoresis result of primer of four-wheel;
Fig. 6: show that the 5th takes turns the 5th the agarose gel electrophoresis result that primer is right;
Fig. 7: show that the 6th takes turns the 6th the agarose gel electrophoresis result that primer is right;
Fig. 8: show that the 7th takes turns the 7th the agarose gel electrophoresis result that primer is right;
Fig. 9: show that the 8th takes turns the 8th the agarose gel electrophoresis result that primer is right;
Figure 10: show that the 9th takes turns the 9th the agarose gel electrophoresis result that primer is right;
Figure 11: show that the tenth takes turns the tenth the agarose gel electrophoresis result that primer is right.
Specific embodiments
In order fully to disclose simple agarose gel electrophoresis method for detecting fast of the present invention, the present invention is described in further detail by the following examples:
1. the extraction of DNA of plants sample: the plant of being adopted in the invention is the Chinese sorghum RIL segregating population of Gramineae sorghum, extracts total DNA that each strain is a blade with the improved CTAB method small-sample method.
2.PCR the setting of system: PCR system 15 μ l, reaction solution consist of 10 * Buffer, 1.5 μ l, MgCl
2(25mmol/L) 0.9 μ l, dNTP (10mmol/L) 1.2 μ l, masterplate DNA (20ng/ μ l) 2.5 μ l, each 0.2 μ l of the forward and reverse primer of Chinese sorghum SSR (10 μ mol/L), Taq archaeal dna polymerase (5U/ μ l) 0.1 μ l (TaKaRa production), sterilization ddH
2O 8.4 μ l.
3.PCR the setting of condition and operation: the PCR response procedures is: 94 ℃ of sex change 30s of 94 ℃ of pre-sex change 5min, and 55 ℃ of annealing 30s, 72 ℃ are extended 45s; 35 circulations; Last 72 ℃ are extended 10min, 4 ℃ of preservations.
4.PCR the detection of product: use agarose gel electrophoresis method for detecting of the present invention that the PCR product is detected.
Agarose gel electrophoresis method for detecting involved in the present invention is that used sepharose is further run glue, washing, dyeing processing, and the recycling gel realizes that its concrete operation method is as follows:
(1) glue: be ready to rubber moulding, the specification 25 * 20cm of gel pallet, the equidistant parallel four row's combs of inserting.Take by weighing 7.5g agarose powder to the erlenmeyer flask of 500ml, add in 250ml 0.5 * TBE electrophoresis buffered soln, shake up.In microwave oven, be heated to agarose and be melted into colloidal sol fully, be cooled to below 60 ℃, in bottle, add fully mixing of 50 μ l EB (final concentration 0.5 μ g/ml) again, pour colloidal sol into rubber moulding, promptly become gel after solidifying.
(2) electrophoresis: carefully extract the comb that is inserted in the gel, gel is put into electrophoresis chamber, add 0.5 * TBE electrophoresis buffered soln to liquid level and cover gel 1-2mm.Draw the PCR product that contains sample-loading buffer of 4.5 μ l and carefully add the comb hole with moving the liquid volley of rifle fire, treat on all samples to connect power supply behind the intact sample, regulating voltage is to 4-5V/cm, and nucleic acid molecule moves on to positive pole from negative pole.When treating that sample-loading buffer is gone to correct position, stop electrophoresis, need 45-60min approximately.
(3) detect: the sepharose that electrophoresis is finished places in the middle of the platform of gel automated imaging system, opens UV-light, can see the DNA band that sends fluorescence, observes and preserve image in computer, and the first run detects and finishes.
(4) washing: the gel that will write down image cuts into rectangular (25 * 4.5cm) of four equalizations along the direction parallel with the point sample hole with gel with the minor operation cutter above the point sample hole, note keeping the integrity in point sample hole, parallel equidistant is in being placed on electrophoresis chamber, connecting power supply runs out of the DNA sample outside the glue, gel is washed, to remove electrophoretic interference next time.
(5) dye glue: the gel after will having washed is immersed in and contains in the electrophoresis buffered soln that final concentration is 0.5 μ g/ml EB 2 hours, promptly can be used for the detection to next molecule marker PCR product, so moves in circles.
5. agarose gel electrophoresis detected result: Fig. 2 has shown electrophoretic result of the first round; Fig. 3-11 shown use successively through after the above-mentioned processing gel carried out second takes turns to the tenth result who takes turns agarose gel electrophoresis.Empirical tests, a clotting glue can reuse 10 times at least, can guarantee that still electrophoretic band is clear, resolving power height, good reproducibility.
Claims (5)
- One kind simple fast, be applicable to the agarose gel electrophoresis method for detecting of high-throughput plant SSR marker site heritable variation rapid detection, described method comprises the step that the gel after detecting through agarose gel electrophoresis is further handled, specific as follows, after last round of detection, the gel that has write down image is placed in the electrophoresis chamber, connect power supply the DNA sample is run out of outside the glue, gel is washed, to remove interference the next round electrophoresis detection; Dye the glue step then, the gel after having washed is immersed in contains in the electrophoresis buffered soln that final concentration is 0.5 μ g/ml EB 2 hours, described gel promptly can be used for the detection of next round to next molecule marker PCR product through after the above-mentioned processing.
- 2. according to the described method of claim 1, for easy to operate, with the gel that write down image with minor operation cutter rectangular about the 25 * 4.5cm that along the direction parallel gel is cut into four equalizations above the point sample hole with the point sample hole, the integrity that note to keep the point sample hole, with its parallel equidistant in being placed on electrophoresis chamber.
- 3. according to the described method of claim 1, wherein said agarose gel electrophoresis detects and comprises glue, electrophoresis, detect step, the glue process is as follows, be ready to rubber moulding, specification 25 * the 20cm of gel pallet, the equidistant parallel four row's combs of inserting, take by weighing 7.5g agarose powder to the erlenmeyer flask of 500ml, add in 250ml 0.5 * TBE electrophoresis buffered soln, shake up, in microwave oven, be heated to agarose and be melted into colloidal sol fully, be cooled to below 60 ℃, adding 50 μ l final concentrations again in bottle is the EB of 0.5 μ g/ml, fully mixing, pour colloidal sol into rubber moulding, promptly become gel after solidifying; Electrophoresis process is as follows, carefully extract the comb that is inserted in the gel, gel is put into electrophoresis chamber, add 0.5 * TBE electrophoresis buffered soln to liquid level and cover gel 1-2mm, draw the PCR product that contains sample-loading buffer of 4.5 μ l and carefully add the comb hole with moving the liquid volley of rifle fire, after treating to finish sample on all samples, connect power supply, regulating voltage is to 4-5V/cm, nucleic acid molecule moves on to positive pole from negative pole, when treating that sample-loading buffer is gone to correct position, stop electrophoresis, need 45-60min approximately; Testing process is as follows, and the sepharose that electrophoresis is finished places in the middle of the platform of gel automated imaging system, opens UV-light, can see the DNA band that sends fluorescence, observes and preserve image in computer.
- 4. according to the described method of claim 1, gel can recycle through after the described processing, and a clotting glue can reuse 10 times at least, can guarantee that still electrophoretic band is clear, resolving power height, good reproducibility.
- 5. according to each described method of claim 1-4, when being used for high-throughput plant SSR marker site heritable variation rapid detection, further comprise extract that plant genome DNA is standby, molecule marker and utilize these primers the genomic dna of Different Individual in different groups or the colony to be carried out the step of pcr amplification in exploitation and synthetic this plant.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899520A (en) * | 2010-07-28 | 2010-12-01 | 中国林业科学研究院热带林业研究所 | Fluorescent dUTP-based method for automatically detecting SSR molecular marker |
| CN107254523A (en) * | 2017-06-14 | 2017-10-17 | 贵州茅台酒股份有限公司 | A kind of glutinous sorghum of making wine routinely plants kind or the method for germplasm Purity |
| CN110935403A (en) * | 2019-12-07 | 2020-03-31 | 云南省农业科学院生物技术与种质资源研究所 | Method for efficiently preparing agarose gel |
| CN115154361A (en) * | 2022-06-22 | 2022-10-11 | 辽宁德玛生物科技有限公司 | Medical and American agarose gel and processing technology thereof |
| CN115440314A (en) * | 2022-09-06 | 2022-12-06 | 湖南艾科瑞生物工程有限公司 | Agarose gel electrophoresis performance detection method and related equipment |
-
2009
- 2009-08-05 CN CN200910160394A patent/CN101671731A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899520A (en) * | 2010-07-28 | 2010-12-01 | 中国林业科学研究院热带林业研究所 | Fluorescent dUTP-based method for automatically detecting SSR molecular marker |
| CN101899520B (en) * | 2010-07-28 | 2013-01-02 | 中国林业科学研究院热带林业研究所 | Fluorescent dUTP-based method for automatically detecting SSR molecular marker |
| CN107254523A (en) * | 2017-06-14 | 2017-10-17 | 贵州茅台酒股份有限公司 | A kind of glutinous sorghum of making wine routinely plants kind or the method for germplasm Purity |
| CN107254523B (en) * | 2017-06-14 | 2020-06-23 | 贵州茅台酒股份有限公司 | Method for identifying conventional variety or germplasm purity of brewing glutinous sorghum |
| CN110935403A (en) * | 2019-12-07 | 2020-03-31 | 云南省农业科学院生物技术与种质资源研究所 | Method for efficiently preparing agarose gel |
| CN115154361A (en) * | 2022-06-22 | 2022-10-11 | 辽宁德玛生物科技有限公司 | Medical and American agarose gel and processing technology thereof |
| CN115440314A (en) * | 2022-09-06 | 2022-12-06 | 湖南艾科瑞生物工程有限公司 | Agarose gel electrophoresis performance detection method and related equipment |
| CN115440314B (en) * | 2022-09-06 | 2023-08-15 | 湖南艾科瑞生物工程有限公司 | Agarose gel electrophoresis performance detection method and related equipment |
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