CN100497623C - Method for extracting whole genome of abyssal sediment - Google Patents
Method for extracting whole genome of abyssal sediment Download PDFInfo
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- CN100497623C CN100497623C CNB2006101368336A CN200610136833A CN100497623C CN 100497623 C CN100497623 C CN 100497623C CN B2006101368336 A CNB2006101368336 A CN B2006101368336A CN 200610136833 A CN200610136833 A CN 200610136833A CN 100497623 C CN100497623 C CN 100497623C
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Abstract
The invention discloses an extracting method of deep-sea sediment genome, which is characterized by the following: disposing sample; extracting DNA; collecting deep-sea sediment sample through aseptic artificial basic salt; adding high-density cell cracking liquid in the experiment sample; cracking cell in the boiling bath; adding the composite liquid of phenol, chloroform and isoamyl alcohol to extract repeatingly; collecting supernatant; sedimenting supernatant through anhydrous alcohol; centrifuging to collect sediment; washing sediment through alcohol; drying sediment in the vacuum; dissolving sediment as extract of genome DNA.
Description
Technical field the present invention relates to the extraction of Deep-Sea Microorganisms DNA, is specifically related to the extraction of pelagic deposit sample gene group, belongs to the marine biotechnology field.
Background technology Deep-Sea Microorganisms genetic resources and enzyme resource industries are called as the sunrise industry of the blue ocean of 21 century Sustainable development, thereby the Deep-Sea Microorganisms The Study on Resources has become current scientific research focus.At present, Protocols in Molecular Biology is the core technology of understanding and exploitation abyssopelagic organism circle Microbial resources, and the genomic dna of acquisition certain mass and concentration is the basis of carrying out molecular biology research.Genome DNA extraction method at microorganism in land or the fresh water environment emerges in an endless stream in recent years, but particularly the method for the genome DNA extraction of Deep-Sea Microorganisms is comparatively deficient at marine microorganism.At present, method of microorganism comes the genome of extracting Deep-Sea Microorganisms in scientific workers extracting land commonly used or the fresh water environment, but unique living environments such as deep-sea high pressure, high temperature or low temperature, oxygen and nutritive substance scarcity must influence form and the structure of microorganism, as form thick cell walls and pod membrane etc., these structural changess will influence the application ordinary method and obtain its genome, hinder further research.In addition because the deep-sea sample collecting is very difficult, thereby as how minimum sample to obtain maximum Microbial resources and genetic resourceses also be the problem that the marine organisms scholar must solve.
Summary of the invention the purpose of this invention is to provide a kind of method of efficient, economic, simple and easy handling, in order to extract high-quality Deep-Sea Microorganisms complete genome DNA from all kinds of samples such as pure growth of pelagic deposit sample, deep sea liquid or solid enriched substance, deep-sea yeast or deep-sea bacterium.
The present invention makes full use of suitable high temperature not only can all microbial cell walls of cracking, and can make DNA and protein denaturation; According to the characteristic of DNA self recoverability and cohesion in suitable temperature, and protein can quicken sex change and precipitation under high temperature and high salt condition, thereby cellular constituents such as DNA and protein are separated.
For achieving the above object, the present invention adopts following technical scheme:
The total genomic method for extracting of abyssal sediment comprises:
1) processing of sample: with the pelagic deposit sample that the basic salt dissolving of aseptic artificial seawater is collected, collect supernatant liquor behind the mixing,, collect supernatant liquor to the washing of settling redissolve; Supernatant liquor places the whizzer high speed centrifugal, collects particulate matters such as thalline; Thalline is with ditalimfos phthalate buffer washing, the centrifugal impurity of removing, and the settling of collecting is a laboratory sample.
2) extracting of DNA: 1. add the high density cell pyrolysis liquid in laboratory sample, fully mixing is put into boiling water bath 8-15min; If in the laboratory sample with Gram-negative bacteria when in the majority: the boiling water bath time generally is controlled in the 10min, if the microorganism of the numerous particulate matter of the outer absorption of pod membrane or polysaccharide or born of the same parents is mainly arranged outward with gram-positive microorganism or cell walls in the laboratory sample, the boiling water bath time preferably is controlled at 10-15min; 2. change in the 55-62 ℃ of water-bath more than the water-bath 30min.3. change over to subsequently in 72 ℃ of water-baths more than the water-bath 30min.4. add the mixed liquid of phenol/chloroform/primary isoamyl alcohol, mixing at room temperature leaves standstill about 10min, and is centrifugal, and supernatant liquor is repeated extracting once with phenol/chloroform/primary isoamyl alcohol, collects supernatant liquor.5. supernatant liquor uses-20 ℃ to 4 ℃ dehydrated alcohol to precipitate, and precipitation temperature is 4 ℃, more than the sedimentation time 30min, and centrifugal collecting precipitation.6. be twice of 70% washing with alcohol throw out with concentration.7. throw out places room temperature or vacuum-drying, with TE or aseptic deionized water dissolution precipitation, is extracting genome DNA liquid.
The final concentration of each composition of described high density cell pyrolysis liquid is: 0.10mol/l≤EDTA≤0.30mol/l; 0.005mol/l≤TrisHCl≤0.150mol/l; 0.10mol/l≤NaCl (mol/l)≤0.30mol/l; The mass percentage concentration of 2%≤SDS≤7.5%; The mass percentage concentration of 0.30%≤CTAB≤0.75%.
The present invention uses the principle of microbiology, biological chemistry, molecular biology, physics and chemistry, the extracting method of a kind of DNA is provided, to solve problems such as the efficient that exists in the total genomic dna leaching process at the special Deep-Sea Microorganisms in habitat is low, versatility is poor, productive rate is low, cost is high, the sample consumption is big, thereby improve productive rate and the kind quantity of DNA in the sample of deep-sea, so that obtain the kind of more Deep-Sea Microorganisms genetic resources and the more Deep-Sea Microorganisms of understanding, make its molecular ecology result of study more comprehensively, more accurate.
Pelagic deposit sample process for extracting complete genome DNA of the present invention is different from methods such as traditional freeze grinding-enzymolysis, CTAB-enzymolysis, its advantage be make full use of under suitable high temperature and high salt condition not only can disruption of microorganisms cell walls and cytolemma, and can make protein denaturation and precipitation; By different thermograde change process, to remove the pollution of materials such as protein, carbohydrate and cell wall fragments.Present method not only can be used for the total genomic extracting of pelagic deposit sample, but also can be used for the genome extracting of deep-sea yeast and some mining bacteriums.The good concentration height of genome quality that obtains can be widely used in every researchs such as microorganism molecule ecological diversity, Molecular Identification, goal gene amplification, phyletic evolution.
Be described in further detail the present invention below in conjunction with accompanying drawing:
Description of drawings
The sample gene group electrophoresis picture that Fig. 1 arrives with this method extracting.Wherein: 1 represents negative control; 2 genomes of representing extracting deep-sea Rhodosporidium diobovatum to obtain, 3 total genomes of representing extracting pelagic deposit sample to obtain; 4 total genomes of representing extracting mineral water sample to obtain; Marker is lambda DNA-HindIII digest marker (1ug); The size of numeral DNA shown in the arrow, unit is kb.
The genomic 18S rDNA that obtains of deep-sea Rhodosporidium diobovatum that Fig. 2 pcr amplification obtains.Wherein 1 represent negative control; Marker is 100bp marker; The 18S rDNA sequence that is about 1800bp that 2 representatives obtain; The size of numeral DNA shown in the arrow, unit is bp.
The genomic 16rDNA that obtains of the pelagic deposit sample that Fig. 3 pcr amplification obtains.Wherein 1 represent the 16rDNA sequence that is about 1500bp that obtains; 2 represent negative control; 3 represent positive control; Marker is 100bp marker; The size of numeral DNA shown in the arrow, unit is bp.
Species diversity rflp analysis finger printing in Fig. 4 pelagic deposit sample.Wherein: M represents 100bp marker; The size of numeral DNA shown in the arrow, unit is bp; On behalf of different clone's, the electrophoretogram of Marker central authorities cut the finger printing that obtains through enzyme.
The genomic ITS1 sequence that obtains of deep-sea Rhodosporidium diobovatum that Fig. 5 pcr amplification obtains.Wherein, the ITS1 sequence that is about 600bp of 1 representative acquisition; 2 represent negative control; M represents 100bp marker; The size of numeral DNA shown in the arrow, unit is bp.
Embodiment
Embodiment 1: total genomic extracting in the deep-sea ooze sample
(1) under aseptic condition, take by weighing deep-sea ooze sample 3g and move in the centrifuge tube aseptic with cover that volume is 50ml, add the basic salt of the aseptic artificial seawater of 30ml, fully vibration is placed on the shaking table and at the uniform velocity shakes 10-20min with 180rpm speed, the centrifugal 3min of 3000rpm then, supernatant liquor is transferred in the new centrifuge tube; Precipitate repeated washing twice again, all collect supernatant liquor, precipitation is preserved standby.
(2) supernatant liquor is with the centrifugal 15min of 12000rpm, collecting precipitation.
(3) add 20ml ditalimfos phthalate buffer in precipitation, fully vibration is placed on the shaking table and at the uniform velocity shakes 10min with 180rpm speed, with the centrifugal 15min of 12000rpm, collecting precipitation.
(4) repeating step is (3) twice.Precipitation is laboratory sample.Getting micro-laboratory sample carries out the microscopic morphology observation and carries out gramstaining, the form of microorganism in the preliminary observation sample.
(5) laboratory sample is transferred in the 5ml centrifuge tube fully, add high density cell pyrolysis liquid (according to 1g:10ml), abundant vibration 10min, (if laboratory sample is with Gram-negative bacteria when in the majority: the boiling water bath time generally is controlled in the 10min to put into the boiling water bath and the 8-15min that picks up counting immediately; If the microorganism of the numerous particulate matter of the outer absorption of pod membrane or polysaccharide or born of the same parents is mainly arranged outward with gram-positive microorganism or cell walls in the laboratory sample, the boiling water bath time preferably is controlled at 10-15min), every overturn gently centrifuge tube 5-8 time of 2-3min.
(6) then change water-bath 1h in the 55-60 ℃ of water-bath over to, every overturn gently centrifuge tube 5-8 time of 15min.
(7) change water-bath 1h in 72 ℃ of water-baths subsequently over to, every overturn gently centrifuge tube 5-8 time of 15min.
(8) in centrifuge tube, add isopyknic phenol/chloroform/primary isoamyl alcohol (V/V/V:25:24:1), reverse gently up and down behind the centrifuge tube 15min, room temperature (25 ℃) leaves standstill 10min subsequently, and the then centrifugal 5min of 12000rpm collects supernatant liquor gently and is transferred in the new centrifuge tube.
(9) repeating step (8) once.
(10) add-20 ℃ of refrigerated dehydrated alcohols of 2.5 times of volumes in the centrifuge tube, reverse gently up and down behind the centrifuge tube 10 times, place 4 ℃ of refrigerators to precipitate 1h at least.The centrifugal 15min of 12000rpm after precipitation finishes, collecting precipitation.
(11) add the ethanol of 2ml70% in being loaded with sedimentary centrifuge tube, reverse gently and leave standstill 15min behind the solution 8 times, the centrifugal 15min of 12000rpm abandons supernatant, collecting precipitation subsequently.
(12) repeating step (11) once.
(13) after throw out places room temperature or vacuum-drying, add an amount of TE or aseptic double-distilled water dissolution precipitation, be extracting genome DNA liquid, 4 ℃ of preservations are standby.
(1) the bacterium liquid 5ml of collection logarithmic phase, the centrifugal 5min of 12000rpm, collecting precipitation.
(2) add 5ml ditalimfos phthalate buffer in precipitation, fully vibration is placed on the shaking table and at the uniform velocity shakes 10min with 180rpm speed, with the centrifugal 15min of 12000rpm, collecting precipitation.
(3) repeating step is (2) twice.Precipitation is laboratory sample.
(4) add high density cell pyrolysis liquid (according to 1g:10ml) in centrifuge tube, the 10min that fully vibrates puts into the boiling water bath and the 10-15min that picks up counting immediately, puts upside down the EP pipe gently 5-8 time every 2-3min.Other step is with embodiment 1.
The component (g/l) of the basic salt of described artificial seawater is: NaCl:24.4770; Na
2SO
4: 3.9170; KCl:0.6640; MgCl
26H
2O:4.9810; CaCl
2H
2O:1.1020; NaHCO
3: 0.1920; KH
2PO
4: 0.1500; MnCl
24H
2O:0.2000; FeSO
47H
2O:2.0000; Na
2S
2O
4: 1.0000.
The configuration of described high density cell pyrolysis liquid (cumulative volume 100ml): TrisHCl (pH8.0,1mol/l), 12.5ml; EDTA (pH 8.0,0.5mol/l), and 50ml; NaCl (5mol/l), 5ml; SDS (20%), 25ml; CTAB (10%), 7.5ml.
Claims (2)
1. the total genomic method for extracting of abyssal sediment is characterized in that: comprising:
1) processing of sample: with the pelagic deposit sample that the basic salt dissolving of aseptic artificial seawater is collected, collect supernatant liquor behind the mixing,, collect supernatant liquor to the washing of settling redissolve; Supernatant liquor places whizzer centrifugal, collects thalline; Thalline is with ditalimfos phthalate buffer washing, the centrifugal impurity of removing, and the settling of collecting is a sample;
2) extracting of DNA: 1. add the high density cell pyrolysis liquid in sample, fully mixing is put into boiling water bath 8-15min; 2. change in the 55-62 ℃ of water-bath more than the water-bath 30min; 3. change over to subsequently in 72 ℃ of water-baths more than the water-bath 30min; 4. add the mixed liquid of phenol/chloroform/primary isoamyl alcohol, mixing at room temperature leaves standstill 10min, and is centrifugal, repeats extracting, collects supernatant liquor; 5. supernatant liquor uses-20 ℃ to 4 ℃ dehydrated alcohol to precipitate, and precipitation temperature is 4 ℃, more than the sedimentation time 30min, and centrifugal collecting precipitation; 6. be twice of 70% washing with alcohol throw out with concentration; 7. throw out places room temperature or vacuum-drying, with TE or aseptic deionized water dissolution precipitation, is extracting genome DNA liquid;
The component of the basic salt of described artificial seawater is: NaCl:24.4770; Na
2SO
4: 3.9170; KCl:0.6640; MgCl
26H
2O:4.9810; CaCl
2H
2O:1.1020; NaHCO
3: 0.1920; KH
2PO
4: 0.1500; MnCl
24H
2O:0.2000; FeSO
47H
2O:2.0000; Na
2S
2O
4: 1.0000; Wherein the unit of each component is g/l;
The final concentration of each composition of described high density cell pyrolysis liquid is: 0.10mol/l≤EDTA≤0.30mol/l; 0.005mol/l≤TrisHCl≤0.150mol/l; 0.10mol/l≤NaCl≤0.30mol/l; The mass percentage concentration of 2%≤SDS≤7.5%; The mass percentage concentration of 0.30%≤CTAB≤0.75%.
2. method for extracting according to claim 1 is characterized in that: with Gram-negative bacteria when in the majority, the boiling water bath time is controlled in the 10min in the sample; Being mainly gram-positive microorganism or cell walls in the sample has outward when pod membrane or polysaccharide or born of the same parents are outer to adsorb the microorganism of numerous particulate matter, and the boiling water bath time is controlled at 10-15min.
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| CN101148664B (en) * | 2007-09-03 | 2011-06-22 | 湖南大学 | Method for extracting solid-state fermentation microorganism total DNA |
| CN111534509B (en) * | 2020-05-18 | 2022-05-17 | 中国科学院深海科学与工程研究所 | Composition, reagent, kit and application for deep-sea microorganism in-situ cell lysis |
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Non-Patent Citations (2)
| Title |
|---|
| 深海沉积物中微量DNA的提取及应用. 赵晶等.海洋与湖沼，第34卷第3期. 2003 |
| 深海沉积物中微量DNA的提取及应用. 赵晶等.海洋与湖沼，第34卷第3期. 2003 * |
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