CN103773870A - Preparation method for directly applying gramineous forage grass radicle to RAPD (Random amplification polymorphism DNA) reaction and kit thereof - Google Patents

Preparation method for directly applying gramineous forage grass radicle to RAPD (Random amplification polymorphism DNA) reaction and kit thereof Download PDF

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CN103773870A
CN103773870A CN201410023265.3A CN201410023265A CN103773870A CN 103773870 A CN103773870 A CN 103773870A CN 201410023265 A CN201410023265 A CN 201410023265A CN 103773870 A CN103773870 A CN 103773870A
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radicle
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rapd
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刘志鹏
周强
马利超
王彦荣
陈天龙
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Lanzhou University
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Abstract

The invention mainly relates to a method for directly applying gramineous forage grass radicle to an RAPD (Random amplification polymorphism DNA) reaction, and in particular relates to an alkali treatment method for the gramineous forage grass radicle and an amplification system of the RAPD reaction. The method for directly applying the gramineous forage grass radicle to the RAPD reaction is mainly characterized by comprising the following steps: treating the gramineous forage grass radicle by using a 0.25M NaOH aqueous solution at 100 DEG C for 1 minute; carrying out the RAPD reaction by directly taking the treated gramineous forage grass radicle as a PCR (Polymerase Chain Reaction) template. The RAPD reaction amplification system is mainly characterized in that a PCR system is 20 microliters in total volume, and comprises 10 microliters of 2*PCR Master (Sangon product number: SK2082), 2 microliters of primer RAPD1 or primer RAPD2, 8 microliters of ddH2O, and 1-2mm of alkali-treated gramineous forage grass radicle. Proved by results, the method has the characteristics of timing conservation, labor conservation, low cost, high repeatability, rapidness and convenience, and has a wide application prospect in the biotechnical field of pasture in China. Advanced technical support is provided for the rapid development of the scientific and research of pasture in China.

Description

Graminous pasture radicle is directly used in preparation method and the test kit thereof of RAPD reaction
Technical field
The present invention relates generally to a kind of graminous pasture radicle and is directly used in the preparation method that RAPD reacts.
Background technology
RAPD(Random Amplified Polymorphic DNA) be randomly amplified polymorphic DNA mark, be to be based upon PCR(Polymerase Chain Reaction) a kind of molecular engineering that can carry out to the genome of whole unknown nucleotide sequence polymorphism analysis on basis.It is take genomic dna as template, take the Random amplified polymorphic nucleotide sequence (being generally 10 base pairs) of single synthetic as primer, under heat-staple archaeal dna polymerase (Taq enzyme) effect, carries out pcr amplification.Amplified production separates, after ethidium bromide staining, on ultraviolet scenograph, detects polymorphism through agarose or polyacrylamide gel electrophoresis.
The first step of RAPD reaction is exactly to extract the genomic dna of testing sample, and the method for generally extracting plant genome DNA has CTAB(Hexadecyltrimethylammonium Bromide) method, SDS(Sodium Dodecyl Sulfate) method or directly utilize test kit to extract.But these methods all take time and effort, especially will carry out large batch of DNA extraction time, tend to like this delay experiment progress, experimental cost is improved.Therefore, a kind of easy method of carrying out RAPD reaction can play a multiplier effect, forefathers had some explorations in this respect, as Virk etc. has set up micro-satellite analysis system fast and effectively in the plants such as Arabidopis thaliana, and Wang Xiufeng etc. utilizes wheat leaf blade to be directly used in PCR and RAPD reaction, though the method for this quick RAPD reaction has been applied to various plants material, but on herbage, but apply lessly, the method for the quick RAPD of this graminous pasture reaction will expand the range of application of biotechnology on graminous pasture.
Summary of the invention
Object one of the present invention be in order to save time, laborsaving, reduce experimental cost, a kind of preparation method of graminous pasture radicle of the RAPD of being directly used in reaction is proposed.
Object two of the present invention is to provide a kind of graminous pasture radicle and is directly used in the test kit that RAPD reacts.
To fast, efficiently and accurately have the RAPD reaction about graminous pasture.This method is time saving and energy saving, easy and simple to handle, under laboratory condition, in the short period, can be good at.In research, 19 kinds of graminous pastures choosing are at random all applicable, have very strong promotional value, can guarantee normally carrying out of scientific effort, and can improve the conventional efficient in scientific research, for the fast development of China's scientific research cause provides advanced technical support and lays a solid foundation.
For achieving the above object, the technical scheme that the present invention takes is: a kind of preparation method of graminous pasture radicle of the RAPD of being directly used in reaction, its principal feature is to comprise the steps:
A. collect seed, adopt double-deck filter paper to sprout method, sprout 65～80h at 23～27 ℃, cut the long radicle of 1～3cm
The tip of a root is for subsequent use;
B. the alkaline purification to graminous pasture radicle; Cut the long radicle of 1～2cm, put into the Eppendorf pipe containing 350～450 μ l0.25mol/LNaOH, this centrifuge tube is inserted in boiling water bath and boils 1～1.5min, after taking-up, add 350～450 μ l0.25mol/L HCl and 200 μ l0.5mol/L Tris-HCl (PH8.0), again this pipe is put into boiling water and hatch 3min, finally choose 1～2mm, directly carry out RAPD reaction as pcr template through the radicle for examination herbage of above-mentioned alkaline purification.
Graminous pasture radicle is directly used in a test kit for RAPD reaction, it is characterized in that including: 2 × PCRMaster, primer RAPD1:CACGGCACAA or primer RAPD2:ACGGCGTATG, ddH 2o, the alkali-treated graminous pasture radicle tip of a root.
Graminous pasture radicle is directly used in a method for RAPD reaction, and its step includes:
A. collect seed, adopt double-deck filter paper to sprout method, sprout 65～80h at 23～27 ℃, cut the long radicle tip of a root of 1～3cm for subsequent use;
B. the alkaline purification to graminous pasture radicle; Cut the long radicle of 1～2cm, put into the Eppendorf pipe containing 350～450 μ l0.25mol/LNaOH, this centrifuge tube is inserted in boiling water bath and boils 1～1.5min, after taking-up, add 350～450 μ l0.25mol/L HCl and 200 μ l0.5mol/L Tris-HCl (PH8.0), again this pipe is put into boiling water and hatch 3min, finally choose 1～2mm and directly carry out RAPD reaction as pcr template through the radicle for examination herbage of above-mentioned alkaline purification;
C. the alkali-treated graminous pasture radicle of the 1～2mm tip of a root above-mentioned B step being obtained carries out pcr amplification, PCR cumulative volume is 20 μ l, comprising 2 × PCR Master10 μ l, 2 μ l primer RAPD1:CACGGCACAA or primer RAPD2:ACGGCGTATG, ddH 2o8 μ l, pcr amplification condition is: (1) 95 ℃ of denaturation 5min; (2) 94 ℃ of sex change 1min; (3) 36 ℃ of annealing 2min; (4) 72 ℃ are extended 1min; (5) 2～4 step cycle 45 times; (6) 72 ℃ are extended 10min; (7) 4 ℃ of preservations;
D. amplified production separates, after ethidium bromide staining, on ultraviolet scenograph, detects polymorphism through agarose or polyacrylamide gel electrophoresis.
The invention has the beneficial effects as follows, can effectively improve conventional efficient, when scientific research personnel can replace CTAB method, the SDS method of general use in the time carrying out large batch of DNA extraction, reach save time, labour-saving effect.The method have save time, laborsaving, cost is low, the features such as the strong and rapid and convenient of repeatability, in the scientific research field of China's biotechnology class, have broad application prospects, can guarantee normally carrying out of scientific effort, and can improve the conventional efficient in scientific research, for the fast development of China's scientific research cause provides advanced technical support and lays a solid foundation.
Accompanying drawing explanation
The primer RAPD1 amplification collection of illustrative plates of Fig. 1 .19 kind graminous pasture radicle.In figure: M represents DL2000 DNAMarker, 1～19 represents the numbering of 19 kinds of graminous pastures, and CK represents the contrast that water is cooked;
The primer RAPD2 amplification collection of illustrative plates of Fig. 2 .19 kind graminous pasture radicle.In figure: M represents DL2000 DNAMarker, 1～19 represents the numbering of 19 kinds of graminous pastures, and CK represents the contrast that water is cooked;
The primer RAPD1 amplification collection of illustrative plates of Fig. 3 .19 kind graminous pasture genomic dna.In figure: M represents DL2000DNA Marker, 1～19 represents the numbering of 19 kinds of graminous pastures, and CK represents the contrast that water is cooked;
The primer RAPD2 amplification collection of illustrative plates of Fig. 4 .19 kind graminous pasture genomic dna.In figure: M represents DL2000DNA Marker, 1～19 represents the numbering of 19 kinds of graminous pastures, and CK represents the contrast that water is cooked;
Fig. 5 .6 kind graminous pasture passes through the result comparison after ethidium bromide staining through radicle and the contrast of alkaline purification.In figure: CK represents the radicle without alkaline purification, alkaline purification represents the radicle through alkaline purification, and light field is illustrated in white light
The effect of taking pictures under condition, UV-light is illustrated in the effect of taking pictures under uv irradiating.
Embodiment
Below in conjunction with embodiment, principle of the present invention and feature are described, example, only for explaining the present invention, is not intended to limit scope of the present invention.
Embodiment 1: a kind of preparation method of the graminous pasture radicle that is directly used in RAPD reaction, comprises the steps:
A. collect seed, adopt double-deck filter paper to sprout method, sprout 65～80h at 23～27 ℃, cut the long radicle root of 1～3cm
Point just carries out alkaline purification to it;
B. the alkaline purification to graminous pasture radicle; Cut the long radicle of 1～2cm, put into the Eppendorf pipe containing 350～450 μ l0.25mol/LNaOH, this centrifuge tube is inserted in boiling water bath and boils 1～1.5min, after taking-up, add 350～450 μ l0.25mol/L HCl and 200 μ l0.5mol/L Tris-HCl (PH8.0), again this pipe is put into boiling water and hatch 3min, finally choose 1～2mm, directly carry out RAPD reaction as pcr template through the radicle for examination herbage of above-mentioned alkaline purification.
Embodiment 2: a kind of graminous pasture radicle is directly used in the test kit of RAPD reaction, it is characterized in that including:
PCR cumulative volume is 20 μ l, numbers: SK2082) 10 μ l, 2 μ l primer RAPD1:CACGGCACAA or primer RAPD2:ACGGCGTATG, ddH comprising the raw chemical product in 2 × PCR Master(Shanghai 2o8 μ l, the alkali-treated graminous pasture radicle of the 1～2mm tip of a root.
Embodiment 3: a kind of graminous pasture radicle is directly used in the method for RAPD reaction, and its step includes:
A. collect 19 kinds of different graminous pasture seeds (in table 1), adopt double-deck filter paper to sprout method, 25 ℃ of sproutings,
While being longer than 2cm Deng the radicle of seed, just it is carried out to alkaline purification;
B. the alkaline purification to graminous pasture radicle; Cut the long radicle of 1～2cm, put into the Eppendorf pipe containing 350～450 μ l0.25mol/LNaOH, this centrifuge tube is inserted in boiling water bath and boils 1～1.5min, after taking-up, add 350～450 μ l0.25mol/L HCl and 200 μ l0.5mol/L Tris-HCl (PH8.0), again this pipe is put into boiling water and hatch 3min, finally choose 1～2mm and directly carry out RAPD reaction as pcr template through the radicle for examination herbage of above-mentioned alkaline purification;
C. the alkali-treated graminous pasture radicle of the 1～2mm tip of a root above-mentioned B step being obtained carries out pcr amplification, PCR cumulative volume is 20 μ l, number comprising the raw chemical product in 2 × PCR Master(Shanghai: SK2082) 10 μ l, 2 μ l primer RAPD1:CACGGCACAA or primer RAPD2:ACGGCGTATG, ddH 2o8 μ l, pcr amplification condition is: (1) 95 ℃ of denaturation 5min; (2) 94 ℃ of sex change 1min; (3) 36 ℃ of annealing 2min; (4) 72 ℃ are extended 1min; (5) 2～4 step cycle 45 times; (6) 72 ℃ are extended 10min; (7) 4 ℃ of preservations;
D. the PCR product having increased is swum with 2% sepharose leakage of electricity, the voltage of general available 120V runs glue 21min;
E. run glue and utilize gel imaging instrument to take pictures to it after completing, see Fig. 1 and Fig. 2.
The essential information of table 119 kind of graminous pasture
Figure BDA0000458428910000051
Embodiment 4: the method for carrying out RAPD reaction as pcr template with the graminous pasture genomic dna extracting, comprises the steps:
In order to prove more fully simple and direct property and the feasibility of this method, compare by the template that the graminous pasture genomic dna extracting (in examination plant and embodiment 3 being same plant) is made pcr amplification as the template graminous pasture radicle next and that processed of pcr amplification.
Extracting routinely the genomic dna of 19 kinds of different graminous pastures (table 1), is 50ng/ μ l by its concentration dilution, as template, adopts RAPD1 and RAPD2 primer to carry out respectively pcr amplification.
PCR system is: the raw chemical product numbering in 2 × PCR Master(Shanghai: SK2082) 10 μ l, 2 μ l primer primer RAPD1:CACGGCACAA or primer RAPD2:ACGGCGTATG, ddH 2o6 μ l, DNA2 μ l.Pcr amplification condition is: (1) 95 ℃ of denaturation 5min; (2) 94 ℃ of sex change 1min; (3) 36 ℃ of annealing 2min; (4) 72 ℃ are extended 1min; (5) 2～4 step cycle 45 times; (6) 72 ℃ are extended 10min; (7) 4 ℃ of preservations.
PCR product detects its effect through 2% agarose gel electrophoresis, see Fig. 3, Fig. 4, result shows, electrophoretic band (Fig. 3 and Fig. 4) using 19 kinds of different graminous pasture genomic dnas as the template graminous pasture radicle different from 19 kinds that pass through alkaline purification is basic identical as the electrophoretic band (Fig. 1, Fig. 2) of template.Although both have certain difference in the brightness of band, but this species diversity can't affect experimental result, within the scope of certain error, can replace graminous pasture genomic dna as the template in RAPD reaction with the graminous pasture radicle of process alkaline purification for this reason.
Embodiment 5: the method for the Fluirescence observation that tries graminous pasture radicle through alkaline purification and contrast comprises the steps:
6 kinds of graminous pastures of random choose in embodiment 3, be respectively Gao Dancao, fodder maize, arabian cron, triticale, oat, Elymus nutans, according to the step of embodiment 2, the radicle of graminous pasture is carried out to alkaline purification, then the 30s that the radicle of processing dyeed in ethidium bromide, uses and contrasts without the radicle of alkaline purification.
After having dyeed, alkali-treated radicle is put in gel imaging instrument and is taken a picture with contrast radicle: first under white light, take a picture, then take a picture under UV-light, it the results are shown in Figure 5.
Result shows on alkali-treated radicle, there is the existence of DNA.Its reason is: the cell wall rupture of graminous pasture radicle after alkaline purification, intracellular DNA is spread in radicle surface, therefore the available alkali-treated graminous pasture radicle tip of a root directly carries out RAPD reaction as pcr template.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.

Claims (3)

1. a preparation method who is directly used in the graminous pasture radicle of RAPD reaction, is characterized in that comprising the steps:
A. collect seed, adopt double-deck filter paper to sprout method, sprout 65～80h at 23～27 ℃, cut the long radicle tip of a root of 1～3cm for subsequent use;
B. the alkaline purification to graminous pasture radicle; Cut the long radicle of 1～2cm, put into the Eppendorf pipe containing 350～450 μ l0.25mol/LNaOH, this centrifuge tube is inserted in boiling water bath and boils 1～1.5min, after taking-up, add 350～450 μ l0.25mol/L HCl and 200 μ l0.5mol/L Tris-HCl (PH8.0), again this pipe is put into boiling water and hatch 3min, finally choose 1～2mm, directly carry out RAPD reaction as pcr template through the radicle for examination herbage of above-mentioned alkaline purification.
2. graminous pasture radicle is directly used in a test kit for RAPD reaction, it is characterized in that including:
2 × PCR Master, primer RAPD1:CACGGCACAA or primer RAPD2:ACGGCGTATG, ddH 2o, the alkali-treated graminous pasture radicle tip of a root.
3. graminous pasture radicle is directly used in a method for RAPD reaction, and its step includes:
A. collect seed, adopt double-deck filter paper to sprout method, sprout 65～80h at 23～27 ℃, cut the long radicle tip of a root of 1～3cm for subsequent use;
B. the alkaline purification to graminous pasture radicle; Cut the long radicle of 1～2cm, put into the Eppendorf pipe containing 350～450 μ l0.25mol/LNaOH, this centrifuge tube is inserted in boiling water bath and boils 1～1.5min, after taking-up, add 350～450 μ l0.25mol/L HCl and 200 μ l0.5mol/L Tris-HCl (PH8.0), again this pipe is put into boiling water and hatch 3min, finally choose 1～2mm and directly carry out RAPD reaction as pcr template through the radicle for examination herbage of above-mentioned alkaline purification;
C. the alkali-treated graminous pasture radicle of the 1～2mm tip of a root above-mentioned B step being obtained carries out pcr amplification, PCR cumulative volume is 20 μ l, comprising 2 × PCR Master10 μ l, 2 μ l primer RAPD1:CACGGCACAA or primer RAPD2:ACGGCGTATG, ddH 2o8 μ l, pcr amplification condition is: (1) 95 ℃ of denaturation 5min; (2) 94 ℃ of sex change 1min; (3) 36 ℃ of annealing 2min; (4) 72 ℃ are extended 1min; (5) 2～4 step cycle 45 times; (6) 72 ℃ are extended 10min; (7) 4 ℃ of preservations;
D. amplified production separates, after ethidium bromide staining, on ultraviolet scenograph, detects polymorphism through agarose or polyacrylamide gel electrophoresis.
CN201410023265.3A 2014-01-17 2014-01-17 Preparation method for directly applying gramineous forage grass radicle to RAPD (Random amplification polymorphism DNA) reaction and kit thereof Pending CN103773870A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1319676A (en) * 2001-01-09 2001-10-31 安徽省农业科学院 Use of wheat leaf blade in polymerase chain reaction and random amplification length pleiomorphy reaction

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1319676A (en) * 2001-01-09 2001-10-31 安徽省农业科学院 Use of wheat leaf blade in polymerase chain reaction and random amplification length pleiomorphy reaction

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
张晓佩等: "与多花黑麦草株高性状相关的RAPD标记分析", 《福建畜牧兽医》 *
汪秀峰等: "小麦叶片直接用于PCR和RAPD反应的方法", 《遗传》 *

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Application publication date: 20140507