CN102807979A

CN102807979A - Method for extracting and pretreating microorganism deoxyribonucleic acid (DNA) on biological active carbon

Info

Publication number: CN102807979A
Application number: CN2012103006693A
Authority: CN
Inventors: 刘文君; 张明露; 王占朝
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2012-08-22
Filing date: 2012-08-22
Publication date: 2012-12-05

Abstract

The invention discloses a method for extracting and pretreating microorganism deoxyribonucleic acid (DNA) on biological active carbon. The method is characterized in that mixed solution with concentration of 200mM of sodium hydroxide (NaOH) and 2.0 percent by weight of polyvinylpyrrolidone is used as corrosion removal buffering solution to performing the corrosion removal treatment for the active carbon, the active carbon is eluted by ultrasonic for one minute and then is chemically split, and finally the microorganism DNA on the biological active carbon is extracted. The length of the obtained DNA is more than 20Kb and can completely satisfy the analysis requirement of polymerase chain reaction (PRC). Due to the adoption of the method, the problems of difficulty in elution caused by less biomass and compact combination of the carrier particles on the biological active carbon can be solved, unfavorable influence of humic acid on the extraction of the nucleic acid of the microorganism can be reduced, and the microorganism cells can be adequately split. The method is simple and convenient to operate, the yield of DNA is large, and the purity is high.

Description

Microbial DNA extracts pretreatment process on a kind of biological activated carbon

Technical field

The invention belongs to field of environment engineering technology, be specifically related to microbial DNA extraction pretreatment process on a kind of biological activated carbon.

Background technology

Gac is owing to have the good adsorption performance; Stable chemical performance can anti-strong acid and highly basic, can stand water logging, high temperature, also lighter than water; Be porous hydrophobic adsorbent, so be widely used in stink, colourity, the natural and artificial synthetic organic materials of removing in the tap water.The microbial film that is the main body with mikrobe and the outer polymer of born of the same parents thereof that adheres on the gac is the biodegradable major portion of objectionable impurities in the water body.Therefore the health risk that gac biological degradation mechanism and mikrobe leakage caused during identification and analysis activated carbon surface biological community structure was handled for clear and definite drinking water deep with distribution is significant.

Traditional microorganism detection is to be based upon on the separation and Culture basis of mikrobe, and sense cycle is long, complicated operation, and specificity is not strong.A large amount of mikrobes in the environment can't be realized artificial culture simultaneously; Perhaps the change owing to living environment changes original structure of community in culturing process, therefore only depends on existing microorganism culturing technology can not reflect the microbial diversity that belongs to environment all sidedly.So Protocols in Molecular Biology becomes the main means of research biological activated carbon microflora.Yet, because the living weight on the biological activated carbon of processing drinking water is few relatively, and tight with the carrier granule combination, so thalline is difficult to directly elute from gac.On the other hand; The humic acid that adsorbs on the gac is difficult for taking place biological degradation under field conditions (factors); Show multiple physico-chemical properties such as polarity, oxidation-reduction quality, complexing and absorption again; In the DNA of classics leaching process, mikrobe is swept along wherein, stop the infiltration of denaturing agents such as SDS and suppress the activity of N,O-Diacetylmuramidase and Proteinase K etc., influence the cracking of lysis buffer mikrobe with interaction such as clay and metals ion.The xanthohumic acid and the ulmic acid of stripping can be oxidized to quinone after alkaline bleach liquor cleavage liquid is handled simultaneously, can be reduced to phenol again, can cause part DNA to lose with the phenol entering organic phase of oxidation.

Even adopt different methods such as dialysis, IX, density gradient centrifugation, chromatography and electrophoresis that the nucleic acid crude extract is further purified; Still have the close humic acid component of some physico-chemical properties and nucleic acid to coexist with it, thereby the subsequent P CR amplification that quantitatively reaches that has influence on nucleic acid wait technical process.Yet; Traditional DNA extraction and commercially available DNA extraction test kit all can not effectively address the above problem, and therefore how before lysing cell, to adopt pre-treatment and the effective microbe elution process of removing humic acids just to become the key that obtains the high DNA of the big quality of output from biological activated carbon.

Summary of the invention

The object of the invention is to overcome the defective of prior art, and microbial DNA extraction pretreatment process on a kind of biological activated carbon is provided, and all reaches the dna fragmentation that is applicable to the downstream molecules biologic operation to obtain output and purity.

Microbial DNA extracts pretreatment process on a kind of biological activated carbon, and the method comprising the steps of as follows:

(1) biological activated carbon take off corruption: with biological activated carbon with take off rotten damping fluid and mixed than 1: 10 by mass/volume, behind the vortex 2min,, behind the vortex 2min, be 1.0 * 10 again at rotating speed at 50 ℃ of reaction 10min down ⁴Centrifugal 5min under the rpm; Wherein taking off rotten damping fluid is that 200mM, Vinylpyrrolidone polymer (PVP) concentration are the mixing solutions of 2.0wt% for NaOH concentration;

(2) ultrasonic wash-out: get above-mentioned gac after centrifugal, be dissolved in the deionized water of sterilization, water at normal temperature is bathed 20min behind the vortex 2min, carries out the 1min supersound process, gets ultrasonic back suspension liquid then, carries out the DNA extraction purifying.

Beneficial effect of the present invention is: being the NaOH of 200mM and PVP that concentration is 2.0wt% with concentration, to take off rotten ability high as taking off rotten damping fluid, reduced the disadvantageous effect that humic acid extracts microbial nucleic acids; Ultrasonic wash-out is handled gac and has been overcome on the drinking water biological active carbon few and combine the wash-out difficulty that closely causes with carrier granule because of living weight; The present invention has overcome the defective of traditional direct extraction DNA method, and simple to operate, can obtain high yield and highly purified DNA from biological activated carbon.

Description of drawings

Fig. 1 extracts total DNA electrophorogram for 9 kinds of different methods among the embodiment 1;

Wherein each label band is respectively: 1-λ-Hind III digest DNA Marker; 2-4 is respectively the NaOH+PVP mixing solutions and takes off rotten also

supersound process

1,2,5min; 5-7 is respectively NaOH solution and takes off rotten also

supersound process

1,2,5min, and 8-10 is respectively and does not take off rotten also

supersound process

1,2,5min.

Fig. 2 is the PCR electrophorogram of total DNA of 9 kinds of different methods extraction acquisitions among the embodiment 2;

Wherein each label band is respectively: 1-M100 bp DNA Ladder; 2-4 is respectively the NaOH+PVP mixing solutions and takes off rotten also

supersound process

1,2,5min; 5-7 is respectively NaOH solution and takes off rotten also

supersound process

1,2,5min, and 8-10 is respectively and does not take off rotten also

supersound process

1,2,5min.

Embodiment

To further specify the present invention through specific embodiment and accompanying drawing below.

Embodiment 1: relatively difference is taken off rotten damping fluid and is taken off rotten effect

The biological activated carbon sample is taken from waterworks, Beijing.Taking off rotten damping fluid adopts the concentration of different concns one-component to be respectively the NaCl solution of 50mM, 100mM, 200mM respectively; Concentration is respectively the Tris-HCl solution of 50mM, 100mM, 200mM; Concentration is respectively the NaOH solution of 50mM, 100mM, 200mM and the NaOH+PVP mixing solutions of different concns, and concentration combination is respectively: 50mM+0.5wt%, 100mM+1.0wt%, 200mM+2.0wt% remove humic acid in the gac.It is as shown in table 1 to take off rotten effect.

Taking off in the rotten damping fluid of one-component; Taking off the rotten not variation of back supernatant color of handling like NaCl solution and Tris-HCl; Explain that the two does not all possess the rotten ability of significantly taking off; The NaOH strength of solution is the supernatant light brown during greater than 100mM, explains that the above NaOH solution of 100mM shows certain rotten effect of taking off; Different coordination agents are made up it to be taken off rotten effect and can be improved; The rotten ability of taking off of rotten damping fluid of taking off after the combination improves greatly in this example; Particularly handle the back supernatant and become dark-brown, explain that selection can effectively remove humic acid with the NaOH of 200mM and 2.0% PVP as taking off rotten damping fluid through the rotten damping fluid that takes off of the PVP of the NaOH of 200mM and 2.0%.

Table 1 difference is taken off the rotten effect of taking off of rotten damping fluid

Embodiment 2: output and the quality of extracting DNA

The biological activated carbon sample is taken from waterworks, Beijing.Get 9 parts in biological activated carbon sample, take following processing respectively: humic acid is not taken off in (1), directly is dissolved in the deionized water of sterilization, and water at normal temperature is bathed 20min behind the vortex 2min, and the supersound process time is respectively 1min, 2min, 5min; (2) be that the NaOH solution of 100mM takes off corruption for taking off rotten damping fluid with concentration, the supersound process time is respectively 1min, 2min, 5min; (3) be that the NaOH of 200mM, the PVP of 2.0wt% take off corruption for taking off rotten damping fluid with concentration, the supersound process time is respectively 1min, 2min, 5min processing, extracts DNA then.Respectively get the DNA 4ul that obtains, with 1.0% added the GoldViwe staining agent sepharose carry out electrophoresis, and the electrophoretic image of gained nucleic acid is analyzed with the Bio-Rad gel imaging system, as shown in Figure 1.Through concentration is that NaOH solution and the 200mMNaOH of 100mM, the sample that the PVP of 2.0wt% takes off after rotten damping fluid is handled have all obtained the total DNA band of length more than 20Kb; Do not have then not detect corresponding total DNA band, and take off DNA band that rotten damping fluid handles the back sample apparently higher than the sample that only takes off the corruption processing through NaOH through two components through taking off the rotten sample of handling.

The DNA extraction result is as shown in table 2, and the total DNA A260/A280 value that is obtained is all more than 1.6, and explaining among total DNA does not have protein contamination, individual samples have part RNA to pollute in explanation more than 1.8, can add a little RNA enzyme and remove.Through concentration is that the A260/A230 value of the sample total DNA after the PVP mixing solutions of 200mM NaOH, 2.0wt% takes off rotten the processing is apparently higher than the sample that takes off rotten processing through NaOH solution, so the rotten damping fluid that takes off of NaOH+ PVP takes off rotten effect and obviously is better than NaOH solution.Through concentration is that the NaOH of 200mM, the sample total DNA amount that the PVP mixing solutions of 2.0wt% takes off after corruption is handled are taken off corruption apparently higher than the NaOH solution with 100mM, and wherein ultrasonic 1min handles the DNA that obtains of institute amount maximum.It is big to explain that use DNA extraction method provided by the invention can obtain output, the DNA that quality is high.

Table 2 DNA extraction amount

Embodiment 3: the pcr amplification of total DNA detects

The pcr amplification product of the DNA that each method obtained among the embodiment 2 is detected electrophoretic image such as Fig. 2 through 1.2% agarose gel electrophoresis.Can find out that the total DNA that takes off the sample after rotten damping fluid is handled through the NaOH+PVP mixing solutions is that template all can amplify target stripe, comparing with NaOH solution is that to take off the PCR product that rotten damping fluid obtains maximum.Explain and use DNA that method provided by the invention obtains to satisfy the downstream molecules biologic operation.

Claims

1. microbial DNA extracts pretreatment process on the biological activated carbon, it is characterized in that the method comprising the steps of as follows:

(1) biological activated carbon take off corruption: with biological activated carbon with take off rotten damping fluid and mixed than 1: 10 by mass/volume, behind the vortex 2min,, behind the vortex 2min, be 1.0 * 10 again at rotating speed at 50 ℃ of reaction 10min down ⁴Centrifugal 5min under the rpm; Wherein taking off rotten damping fluid is that 200mM, Vinylpyrrolidone polymer concentration are the mixing solutions of 2.0wt% for NaOH concentration;