CN104195133A - Method for quickly extracting trace DNA (deoxyribonucleic acid) of oilseed rape - Google Patents

Abstract

The invention belongs to the technical field of preparation of genome DNA (deoxyribonucleic acid) of oilseed rape and in particular relates to a method for quickly extracting trace DNA of oilseed rape. The method mainly comprises the following steps: adding an extraction buffer into plant organs and/or tissues, and breaking to obtain plant powder; culturing in a water bath with the temperature of 94-100 DEG C for 10 minutes, centrifuging, sucking supernatant liquid to obtain DNA-containing extraction liquid, diluting the extraction liquid, and storing the diluted extraction liquid at -20 DEG C or directly applying the diluted extraction liquid to PCR (polymerase chain reaction). Compared with the conventional CTAB (cetyl trimethyl ammonium bromide) process in the prior art, the method does not have remarkable difference on the aspect of PCR amplification but has remarkable advantages of high speed, simple operating steps, high efficiency, no toxic hazard and the like.

Description

A kind of method of rapid extraction rape minim DNA

Technical field

The present invention relates to plant genome DNA preparing technical field, be specifically related to a kind of method of rapid extraction rape minim DNA.

Background technology

Rape is one of important in the world oil crops, is important food plant oil source, is also important industrial raw material and protein feed source.Rape oil is the important sources of the self-produced edible vegetable oil of China, and in China's oil crops oil offtake, proportion has reached more than 57.2%, and therefore, rape is produced in China's economy, life and occupied very important status

Rape is an allotrtraploid plant, genome relative complex, and along with the development that biotechnology is advanced by leaps and bounds, different kinds of molecules mark has been widely used in the evaluation of verity and the purity of rape variety.The extraction of DNA is the first step of carrying out molecular marker analysis, and its efficiency and quality are very important.At present, the DNA extraction of rape adopts CTAB method conventionally, and its technological step is comparatively complicated, consuming time oversize, and uses and contain the toxic reagents such as phenol, chloroform, need to operate by stink cupboard, is unfavorable for the wide popularization and application at breeding work.For solving the deficiency existing in above-mentioned DNA extraction, the present invention proposes a kind of simple and efficient, cost is lower, the method for the rapid extraction minim DNA of toxicological harmless, the DNA extracting can meet the demand of molecular marker analysis, and compare with traditional DNA extraction method, the effect of labeled analysis does not have marked difference.

Summary of the invention:

A kind of method that the object of this invention is to provide rapid extraction rape minim DNA, the method is compared with existing Method of Plant DNA Extraction (as modified CTAB method), can greatly simplify extraction process, shortening time and cost-saving.

The present invention is achieved through the following technical solutions:

The material that the present invention utilizes is that (product that for example represent are swede type rape A-grade in the first class 177 to the high oleic acid rape of cabbage type, A-grade in the first class 254, referring to Hua Zhong Agriculture University's invention patent mandate, patent No. ZL2009102734352, Granted publication CN101824472B) young leaflet tablet.

A rapid extraction rape minim DNA method, its step is as follows:

(1) gather swede type rape young leaflet tablet 9-10mg and pack 2ml centrifuge tube into, be placed in ice chest;

(2) add 250ul Extraction buffer Buffer and steel ball, in sample grinding machine, grind 3min;

(3) be placed in 94-100 ℃ of water-bath and cultivate 10min, or water-bath cultivates and be no more than 12min, avoid affecting quality;

(4) under 12000rpm, centrifugal 5min;

(5) draw supernatant liquor, or as required, draw at most 50ul supernatant liquor, add ultrapure water dilution 9-10 doubly, in-20 ℃, preserve or get 2-3ul and be directly used in PCR reaction;

Wherein:

The formula of described Extraction buffer Buffer, by the Tris-HCl (pH7.5) of 500ml 1M, 17.55g NaCl and 102.6g sucrose adding distil water be settled to mix after 1L formulated.

The present invention compared with prior art, has the following advantages:

1, the CTAB method of rapid fractionation method involved in the present invention and improvement compares, it has saved the chloroform in ordinary method: primary isoamyl alcohol (volume ratio 24:1) extracting, dehydrated alcohol precipitation DNA and the air-dry and process such as dissolvings again, greatly saved time and the cost of extraction DNA;

2, the DNA that the present invention extracts is by the pcr amplification in later stage, can complete fast screening and the later stage research of rape large group, greatly improved the large group screening efficiency in rape fundamental research and genetic improvement, make the methods simplifications such as rape gene clone, molecular mark, transgenosis detection, rapid, routinize.

Accompanying drawing explanation

Fig. 1: the electrophoresis result swimming lane that is 2 kinds of DNA extraction methods.Description of symbols in figure: M: laboratory self-control 3KB Marker of the present invention; The method for extracting of swimming lane 1,2,3,4 is conventional CTAB method; Swimming lane 5,6,7,8 methods are the rapid fractionation method that the present invention proposes; Swimming lane 1 and 5,2 and 6,3 and 7,4 and 8 is same sample.

Fig. 2: be the agarose gel electrophoresis figure of pcr amplification product actin gene (GenBank accession:AF111812.1).Description of symbols in figure: swimming lane 1～4: the PCR product of improvement CTAB extracting DNA; Swimming lane 5～8: be the PCR product of the quick extracting DNA of the present invention.

Embodiment:

Below in conjunction with the drawings and specific embodiments, the present invention is further illustrated, but be not restriction the present invention.

Embodiment 1: the extracting method of rape DNA is set up

(product that for example represent are swede type rape A-grade in the first class 177 to choose the high oleic acid rape of cabbage type, A-grade in the first class 254, referring to Hua Zhong Agriculture University's invention patent mandate " patent No. ZL2009102734352; Granted publication CN101824472B) young leaflet tablet, use following two kinds of comparison tests that parallel method is extracted respectively rape genomic dna.

1, the conventional extracting method (being improved method of CTAB) of DNA

1, cotyledon DNA extraction technology.On the basis of the conventional DNA extraction method of forefathers (Porebski et al, 1997, Plant molecular biology reporter, 15:8-15), improve, be called for short single extraction method.

Concrete operation step is as follows:

(1) get swede type rape seedling stage and (for example represent the high oleic acid rape of strain cabbage type A-grade in the first class 177, A-grade in the first class 254, referring to Hua Zhong Agriculture University's invention patent mandate " patent No. ZL2009102734352; Granted publication CN101824472B) about young tender leaf agreement that contracts a film or TV play to an actor or actress 0.2g puts into 2ml centrifuge tube, puts into a steel ball simultaneously;

(2) add CTAB extracting solution (the Porebski et al of 250ul 2%, 1997, Plant molecular biology reporter, 15:8-15), on the broken instrument of plant, grind, after blade grinds completely, add 2%CTAB extracting solution to 800ul;

(3) 65 ℃ of water-bath 0.5h, slowly vibrate once every 10min;

(4) the chloroform cooling equivalent volumes 800ul that adds): primary isoamyl alcohol (volume ratio 24:1) mixing solutions, shakes 15min, the centrifugal 10min of 12000rpm slowly on shaking table;

(5) draw supernatant 500ul to 1.5ml centrifuge tube, add the dehydrated alcohol of 1ml ice, slowly shake up ,-20 ℃ of precipitation 30min, the centrifugal 10min of 12000rpm;

(6) outwell supernatant, by 75% ethanol washing and precipitating, the centrifugal 5min of 8000rpm;

(7) outwell scavenging solution, dry DNA in vacuum drier machine; Add 100ulddH ₂o dissolves ,-20 ℃ of preservations, standby.

2, DNA rapid extraction method of the present invention, concrete operation step is as follows:

(1) (product that for example represent are swede type rape A-grade in the first class 177 to gather the high oleic acid rape of cabbage type, A-grade in the first class 254, referring to Hua Zhong Agriculture University's invention patent mandate " patent No. ZL2009102734352; Granted publication CN101824472B) young leaflet tablet 9-10mg packs 2ml centrifuge tube into, is placed in ice chest;

(2) add 250ul Extraction buffer Buffer and steel ball, in sample grinding machine, grind 3min;

(3) be placed in 94-100 ℃ of water-bath and cultivate 10min (water-bath incubation time should not surpass 12min, and surpassing 12min can affect quality);

(4) under 12000rpm, centrifugal 5min;

(5) draw supernatant liquor (as required, can draw 50ul at most), add ddH ₂o dilution 9-10 doubly, preserves or gets 2-3ul for-20 ℃ and be directly used in PCR reaction;

Wherein:

The formula of above-mentioned Extraction buffer Buffer, by the Tris-HCl (pH7.5) of 500ml 1M, 17.55g NaCl and 102.6g sucrose, adding distil water be settled to mix after 1L formulated.

The detection of embodiment 2:DNA quality

(1) agarose gel electrophoresis detects

By the method for sepharose, two kinds of rape leaf complete genome DNAs of carrying are identified, operate as follows: get 4ulDNA sample and mix with the tetrabromophenol sulfonphthalein that 2ul is mixed with fluorescence dye, the homemade 3KB Marker in laboratory of take is reference, with 1% sepharose, detect the size of DNA, and observe and take pictures with gel imaging system, result is as shown in Figure 1.Experimental result shows: the DNA of quick extracting of the present invention is slightly light yellow, and what improvement CTAB extracted is white.As can be seen from Figure 1, all have the RNA of disperse before 2 kinds of method for extracting, reason is not add RNA enzyme purification to process; But near point sample hole and all there is no a band, does not have the pollutions such as protein in the DNA that shows to extract; And there is master tape in the DNA high molecular district that improvement CTAB method is extracted, and all degradeds to some extent of the DNA of extracting fast show with chloroform: primary isoamyl alcohol (volume ratio 24:1) extracting, can effectively suppress the activity of DNA enzyme, and avoid its degraded.

(2) pcr amplification detects

Adopt the synthetic primer for rape actin gene design (forward primer 5'-AGCTGGAGACGGCTAAGAG-3', the reverse primer 5'-GTTGGAAAGTGCTGAGGGA-3') pcr amplification of Wuhan Qing Ke company.13ul reaction system: 3ul DNA profiling, 1.3ul 10x Buffer is (containing MgCl ₂), 0.25ul dNTP (10mmolL ^-1), 0.13ul Taq enzyme (2.5UuL ^-1), the above-mentioned forward primer (10mmolL of 0.2ul ^-1), 0.2ul reverse primer (10mmolL ^-1), 7.92ul ddH ₂o.Response procedures: 94 ℃ of 4min denaturations; 94 ℃ of sex change 30S, 55 ℃ of annealing 30S, 72 ℃ are extended 30S, totally 35 circulations, reaction product detects through 1% agarose gel electrophoresis, and observation is taken pictures, and obtains result as shown in Figure 2.Test-results shows: the rape genomic dna extracting by fast extracting method of the present invention carries out actin gene amplification as template, can amplify object band, and band clear (seeing Fig. 2 swimming lane 1～4), and take the effect that DNA that modified CTAB method extracts is template amplification and there is no difference (seeing Fig. 2 swimming lane 5～8).The pollution that contains RNA in the DNA of presentation of results extracting does not have a significant effect to pcr amplification, and the DNA that fast extracting method provided by the invention extracts can carry out genomic pcr amplification completely, meets the needs of genotype detection.

Embodiment 3: relatively two kinds of uses of extracting DNA method extraction time and reagent

(1) two kind of method is extracted the comparison of DNA required time

Processing under the prerequisite of same sample, the time of CTAB method processing sample is 110min, and rapid fractionation method processing sample required time of the present invention is only 20min, is about 1/5 of the CTAB method treatment time, can be directly used in pcr amplification.Contrast this two kinds of methods, it is fast that the method for DNA rapid extraction of the present invention has speed, and operation steps is simple, the advantage that efficiency is high, and can be used for the Marker Identification of a large amount of population materials in field.

(2) extract the comparison of DNA agents useful for same

As shown in table 1, CTAB improved method extracts in DNA formula and has used 7 kinds of reagent, and containing high toxic agent in phenol, chloroform etc., and must in Fume Hoods, carry out; And fast extracting method of the present invention only contains 3 kinds of reagent, and substantially there is no toxicity, whole experiment is all carried out in the centrifuge tube of 2ml,

Table 1 is extracting and the comparison of modified CTAB method extracting DNA agents useful for same toxicity fast

The present invention had both reduced the cost of experiment, made that operation is simple, had also reduced the harm to HUMAN HEALTH, and the pollution to the air in environment, water body, be conducive to realize the optimum sustainable development of ecotope.

Claims (1)

1. a method for rapid extraction rape minim DNA, is characterized in that following steps:

(1) gather swede type rape young leaflet tablet 9-10mg and pack 2ml centrifuge tube into, be placed in ice chest;

(2) add 250ul Extraction buffer Buffer and steel ball, in sample grinding machine, grind 3min;

(3) be placed in 94-100 ℃ of water-bath and cultivate 10min, or water-bath cultivates and be no more than 12min, avoid affecting quality;

(4) under 12000rpm, centrifugal 5min;

(5) draw supernatant liquor, or as required, draw at most 50ul supernatant liquor, add ultrapure water dilution 9-10 doubly, in-20 ℃, preserve or get 2-3ul and be directly used in PCR reaction;

Wherein:

The formula of described Extraction buffer Buffer, by the Tris-HCl (pH7.5) of 500ml 1M, 17.55g NaCl and 102.6g sucrose adding distil water be settled to mix after 1L formulated.