CN105385680B - For the reagent that DNA and RNA is extracted simultaneously, extracting method and purposes - Google Patents
For the reagent that DNA and RNA is extracted simultaneously, extracting method and purposes Download PDFInfo
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- CN105385680B CN105385680B CN201510982621.9A CN201510982621A CN105385680B CN 105385680 B CN105385680 B CN 105385680B CN 201510982621 A CN201510982621 A CN 201510982621A CN 105385680 B CN105385680 B CN 105385680B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The present invention relates to genetic engineering fields, and in particular to a kind of reagent extracted simultaneously for DNA and RNA, for the DNA and RNA method extracted simultaneously and the purposes of the reagent.The reagent includes natural saccharide compound, heavy metal salt compounds, neutral salt compounds, thiocyanic acid class compound, alcohol compound, organic solvent class compound, phenolic compound, N- dodecane acyl group sarcosine and the salt compounds for adjusting solution acid alkalinity.The present invention extracts reagent to RNA and DNA and is optimized, and strengthens the protection of DNA and RNA, and room temperature in entire extraction process without carrying out cooling processing, and can fully meet the needs of the subsequent sequencing of two generations and three generations's sequencing.Operating method is simple and fast, and sample requirements are few, DNA the and RNA purity and concentration of extraction are high.
Description
Technical field
The present invention relates to genetic engineering fields, and in particular to a kind of reagent extracted simultaneously for DNA and RNA is used for DNA
With the purposes of the RNA method extracted simultaneously and the reagent.
Background technique
RNA is one of living cells and much viruses macromolecular substances, is condensed into list through phosphide key by ribonucleotide
Chain length chain molecule, by phosphoric acid, ribose and base, (A adenine, G guanine, C cytimidine, U urinate a ribonucleic acid molecule
Pyrimidine) it constitutes.RNA is used in the synthesis process of living cells all proteins, and carries the hereditary information of many viruses.
Currently, sequencing is divided into genome sequencing, exon sequencing, the different method such as specific region sequencing is respectively required for
Different processing methods is used to sample, can not achieve and DNA and RNA is extracted using same reagent, and can satisfy subsequent
The needs of sequencing.
Summary of the invention
The problem of the prior art that present invention needs solve is: existing reagent is only used for DNA extraction or RNA is mentioned
It takes, and its is complex for operation step, there is no that the reagent that DNA and RNA is extracted can be used for simultaneously in existing reagent, face simultaneously
In a small amount of sample extract high quality RNA difficulty.
To solve the above-mentioned problems, the present invention provides a kind of for extracting the examination of DNA and RNA in tissue sample simultaneously
Agent, extracting method and purposes.
Specifically, the present invention provides a kind of reagent extracted simultaneously for DNA in sample and RNA,
The reagent includes natural saccharide compound, heavy metal salt compounds, neutral salt compounds, thiocyanic acid class
Close object, alcohol compound, organic solvent class compound, phenolic compound, N- dodecane acyl group sarcosine and for adjusting solution
The salt compounds of pH value.
Preferably, the concentration of each compound is respectively as follows: in the reagent
(1) natural saccharide compound is 0.1-0.4mol/L, preferably 0.2-0.3mol/L；
(2) heavy metal salt compounds are 0.05-0.5mol/L, preferably 0.1-0.2mol/L；
(3) neutral salt compounds 1.0-2.0mol/L, preferably 1.0-1.4mol/L；
(4) thiocyanic acid class compound 0.9-3.0mol/L, preferably 0.9-1.5mol/L；
(5) alcohol compound 4-15% (v/v), preferably 4-6% (v/v)；
(6) organic solvent class compound 1.55-7.5mol/L, preferably 1.55-3.1mol/L；
(7) phenolic compound 35%-65% (v/v), preferably 35%-45% (v/v)；
(8) N- dodecane acyl group sarcosine 0.01-0.07mol/L, preferably 0.03-0.06mol/L；
(9) for adjusting the salt compounds 0.8-2.5mol/L, preferably 0.8-1.2mol/L of solution acid alkalinity.
Preferably, the natural saccharide compound be selected from one or more of trehalose, isotrehalose, neotrehalose with
On；
The heavy metal salt compounds be selected from one or more of silver nitrate, copper sulphate, frerrous chloride, zinc chloride with
On, preferably copper sulphate or silver nitrate；
The neutrality salt compounds are selected from one or more of sodium chloride, potassium chloride, magnesium chloride, calcium chloride or more,
Preferably sodium chloride or potassium chloride；
The organic solvent class compound is selected from one or more of chloroform, acetone, methanol, ethyl alcohol or more, preferably
The composition of chloroform and ethyl alcohol；
The alcohol compound is the alcohols that carbon atom number is 3-4, is selected from glycerol, normal propyl alcohol, isopropanol, positive fourth
It is more than one or more of alcohol, ethylene glycol, preferably glycerol or normal propyl alcohol；
The thiocyanic acid class compound is selected from one or more of guanidine thiocyanate, ammonium thiocyanate or more, preferably sulphur cyanogen
The composition of sour guanidine and ammonium thiocyanate；
The phenolic compound be selected from one of phenol, p-cresol, o-cresol, m-cresol, phloroglucin or
More than several, preferably phenol；
The salt compounds for adjusting solution acid alkalinity are selected from one or more of sodium acetate, sodium citrate or more,
The preferably composition of sodium citrate and sodium acetate.
Preferably, the reagent include trehalose, copper sulphate, sodium chloride, guanidine thiocyanate, ammonium thiocyanate, ethyl alcohol, glycerol,
Chloroform, phenol, N- dodecane acyl group sarcosine, sodium citrate and sodium acetate.
Preferably, the concentration of each component is respectively as follows: in the reagent
Trehalose 0.1-0.4mol/L, preferably 0.2-0.3mol/L；Copper sulphate 0.05-0.5mol/L, preferably 0.1-
0.2mol/L；Sodium chloride 1.0-2.0mol/L, preferably 1.0-1.4mol/L；Guanidine thiocyanate 0.6-2.0mol/L, it is preferably
0.6-1.0mol/L；Ammonium thiocyanate 0.3-1.0mol/L, preferably 0.3-0.5mol/L；Glycerol 4%-15% (v/v), it is preferably
4-6% (v/v)；Chloroform 1.5-5.0mol/L, preferably 1.5-2.0mol/L；Ethyl alcohol 0.05-2.5mol/L, preferably 0.05-
1.1mol/L；It phenol 35%-65% (v/v), is preferably 35-45% (v/v)；N- dodecane acyl group sarcosine 0.01-
0.07mol/L, preferably 0.03-0.06mol/L；Sodium citrate 0.7-1.5mol/L, preferably 0.7-1.0mol/L；Sodium acetate
0.1-1.0mol/L, preferably 0.1-0.2mol/L.
Invention also provides the methods that reagent described in any of the above item is extracted for DNA and RNA, including walk as follows
It is rapid:
(1) reagent described in any of the above item is added in sample, is centrifuged after concussion, is divided into supernatant liquid and subnatant
Body；
(2) wherein, the extraction of RNA is used in supernatant liquid；
(3) lower liquid is used for the extraction of DNA.
Preferably, the concussion time is 5-10min in step (1), centrifugal speed 10000g-15000g, and centrifugation time is
10-20 minutes.
Preferably, room temperature is shaken after isopropanol is added in liquid at the middle and upper levels for step (2), and centrifugation discards liquid；Then it is added
75% ethyl alcohol, room temperature centrifugation, removes liquid, obtains RNA.
Preferably, it is shaken 10-20 minutes after isopropanol being added, concussion speed is 50rpm-200rpm；Centrifugal speed
10000g-15000g, centrifugation time are 10-20 minutes.
Preferably, after 75% ethyl alcohol is added in step (2), centrifugal speed 10000g-15000g, centrifugation time 10-20
Minute.
Preferably, room temperature concussion is centrifuged after dehydrated alcohol is added in step (3) in lower liquid, removes liquid；Then plus
Enter sodium citrate ethanol solution, removes liquid after room temperature concussion centrifugation, obtain DNA.
Preferably, after dehydrated alcohol being added in step (3), the concussion time is 5-10min, and concussion speed is 50rpm-
200rpm；
After sodium citrate ethanol solution is added, the concussion time is 5-10 minutes, and concussion speed is 50rpm-200rpm.
Preferably, the concentration of the sodium citrate in the sodium citrate ethanol solution is 0.05M-0.2M.
The present invention also provides method described in reagent described in any of the above item or any of the above item for extract and/or
Measure the application in the content of the DNA and RNA in sample.
Preferably, the DNA and RNA of the application fetches can be used as genome sequencing, exon sequencing, specific region survey
Sequence.
Preferably, the sample in the application includes fresh pathological tissues, tumor tissues case wax stone, tumor tissues disease
Example paraffin section, whole blood, blood plasma, serum, pleural effusion or fresh biopsy sample.
Reagent provided by the invention is stored at room temperature, and the shelf-life is 1 year.
Agents useful for same of the present invention can be used for measuring it is various need genome sequencing, exon sequencing, specific region survey
The tissue samples of sequence, if while the amounts of tissue samples need after -20 degrees Celsius shred tissue, grind 2-10 when being more than 1g
Minute can.
The beneficial effects of the present invention are:
(1) reagent of the invention can be used for simultaneously the extraction of DNA and RNA in sample, while strengthen DNA's and RNA
Protection, and a small amount of sample can meet extraction and require, and can fully meet the needs of the subsequent sequencing of two generations and three generations's sequencing.
(2) extraction process of the entire reagent of the present invention, room temperature can be completed, without carrying out low-temperature treatment, operating method letter
Single quick, sample requirements are few, DNA the and RNA purity and concentration of extraction are high.
Below with reference to each specific embodiment, the present invention and its advantageous effects are described in detail, in which:
Specific embodiment
As described above, it is an object of the invention to: reagent a kind of while for DNA and RNA extraction is provided, and simultaneously
Extract the method and purposes of DNA and RNA.
Specifically, the present invention provides a kind of reagent extracted simultaneously for DNA in tissue sample and RNA, the examination
Agent includes natural saccharide compound, heavy metal salt compounds, neutral salt compounds, thiocyanic acid class compound, alcohols chemical combination
Object, organic solvent class compound, phenolic compound, N- dodecane acyl group sarcosine and the salt for adjusting solution acid alkalinity
Close object.
Wherein, in the preferred embodiment of the present invention, the reagent includes trehalose, copper sulphate, sodium chloride, sulphur
Cyanic acid guanidine, ammonium thiocyanate, glycerol, ethyl alcohol, chloroform, phenol, N- dodecane acyl group sarcosine, sodium citrate and sodium acetate.
In a kind of specific preferred embodiment of the invention, the reagent includes trehalose, copper sulphate, sodium chloride, sulphur
Cyanic acid guanidine, ammonium thiocyanate, ethyl alcohol, glycerol, chloroform, phenol, N- dodecane acyl group sarcosine, sodium citrate and sodium acetate, it is described
The concentration of each component is respectively as follows: trehalose 0.1-0.4mol/L, copper sulphate 0.05-0.5mol/L, sodium chloride 1.0- in reagent
2.0mol/L, guanidine thiocyanate 0.6-2.0mol/L, ammonium thiocyanate 0.3-1.0mol/L, glycerol 4%-15% (v/v), ethyl alcohol
0.05-2.5mol/L, chloroform 1.5-5.0mol/L, phenol 35%-65% (v/v), N- dodecane acyl group sarcosine 0.01-
0.07mol/L, sodium citrate 0.7-1.5mol/L, sodium acetate 0.1-1.0mol/L.
In the specific preferred embodiment of another kind of the invention, the reagent include trehalose, copper sulphate, sodium chloride,
Guanidine thiocyanate, ammonium thiocyanate, ethyl alcohol, glycerol, chloroform, phenol, N- dodecane acyl group sarcosine, sodium citrate and sodium acetate, institute
The concentration for stating each component in reagent is respectively as follows: trehalose 0.2-0.3mol/L, copper sulphate 0.1-0.2mol/L, sodium chloride 1.0-
1.4mol/L, guanidine thiocyanate 0.6-1.0mol/L, ammonium thiocyanate 0.3-0.5mol/L, glycerol 4-6% (v/v), ethyl alcohol 0.05-
1.1mol/L, chloroform 1.5-2.0mol/L, phenol 35-45% (v/v), N- dodecane acyl group sarcosine 0.03-0.06mol/L,
Sodium citrate 0.7-1.0mol/L, sodium acetate 0.1-0.2mol/L.
The extraction of the RNA of high quality is to carry out the committed step of many molecular biology experiments.And it is high in a small amount of sample
The extraction of the RNA of quality, it is even more important for the sequencing of two generation of modern genetic and three generations's sequencing.The present invention provides one kind for thin
The instant reagent that RNA is extracted in born of the same parents or tissue, while can extract the DNA in sample, greatly meet the sequencing of two generations and three
Generation sequencing industrialization demand.
Reagent of the invention be used as tissue homogenate when can protect the integrality of RNA molecule, while destroy eucaryotic cell structure,
Cell mixing component.After centrifugation, because the density of different material is different in reagent, solution can be divided into organic phase and water phase.Phenol
Class compound effectively makes the protein denaturation in sample with the organic solvent mixing in solution.Wherein phenol and chloroform is mixed
It closes, can especially efficiently reduce the formation of the undissolvable RNA- albumen composition of interface.Moreover, chloroform can be reduced remaining
The amount of phenol in water phase improves the yield of RNA to reduce the degradation amount of RNA.
Under normal conditions, solute is easier to be dissolved in solvent similar with its structure, i.e., similar to mix.A kind of solvent is whole
Solvability depend on its polarity.For example, a kind of stronger solute of polarity (such as urea) be highly soluble in it is more stronger than its polarity
Solvent (such as water), be insoluble in the comparable solvent of its polarity (such as alcohol), be practically insoluble in non-polar solvent (such as chlorine
Imitative, ether) in.Nucleic acid is polarity macromolecular (because it is with negatively charged phosphoric acid backbone), it is soluble in lower layer
In water phase, and organic phase is not dissolved in (water is more stronger than the polarity of phenol).On the contrary, protein is by the electrification of different proportion and not charged
Structural domain composition, generate hydrophily and water repellent region respectively.Water repellent region and phenol interact, and it is heavy to generate protein
It forms sediment, polymer (carbohydrate containing) is gathered in the layering interfaces (usually white flock) of two solvents, or and lipid
It is dissolved in the organic phase of lower layer together.
The PH of solution decides the separation of DNA and RNA in organic phase and water phase.(the PH7- in neutral or weakly alkaline environment
8) phosphodiester bond, in nucleic acid is negatively charged, and at this moment DNA and RNA are present in water phase.When PH drops to 4.8, DNA exists
Mobility in water phase reaches maximum.In this acidic environment, most protein and small DNA (< 10kb) are gathered in
In organic phase, big DNA molecular and a part of protein aggregation are at the layering of organic phase and aqueous phase interface.The acid item of solution
Part can be such that RNA stays in water phase, DNA is made to be moved to organic phase.Acidic environment can also make the reduction of RNase activity.Thiocyanic acid class
Closing object can be used to reduce influence of the nuclease to experiment.
Natural saccharide compound prevents reagent possible DNA or RNA it acts as protection is provided to DNA and RNA
The damage of chain.
Heavy metal salt compounds, it acts as the activity to RNA enzyme and DNA enzymatic to play inhibiting effect, can guarantee to mention
It takes process that can carry out in room temperature, is not necessarily to low-temperature operation.
The present invention will be further described in detail with reference to the specific embodiments.
Reagent used in the embodiment of the present invention and device information are as follows:
The reagent used in the present invention is the common reagent of those skilled in the art, wherein in embodiment and comparative example
Reagent is purchased from SIGMA, and purity is that analysis is pure.Wherein trehalose is by two glucose molecules with 1,1- glycosidic bond structure
At nonreducing sugar, have 3 kinds of isomers i.e. trehalose (α, α), isotrehalose (β, β) and a neotrehalose (α, β), the present invention is real
Applying the trehalose used in example and comparative example is (α, α) type.
The instrument that DNA and RNA concentration mensuration is used is 2000 ultramicrospectrophotometer of Nanodrop, is purchased from Thermo
Scientific。
Wherein, the preparation method of 0.1M sodium citrate ethanol solution is as follows:
29.41g sodium citrate (with the template rolled well) is accurately weighed, is put into water-soluble with distilling in right amount in 200ml beaker
Solution, lysate are transferred to the volumetric flask of 1000ml, 100% ethyl alcohol 10ml are added, with distilled water constant volume to 1000ml.It is transferred to dry
In net reagent bottle, label spare.The preparation side of 0.05M and 0.2M sodium citrate ethanol solution in embodiment two and embodiment three
Method is prepared with reference to the preparation method of 0.1M sodium citrate ethanol solution.
The formula of agents useful for same in each embodiment and comparative example of table 1
Wherein, in table 1 "-" represent be not add corresponding component, i.e. the content of respective components is zero.
Embodiment one
(1) processing of tissue sample
After taking 10mg test serum sample (mouse liver tissue) to be homogenized in liquid nitrogen, it is added as in 1 embodiment one of table
Reagent 1ml, room temperature 200rpm shake after five minutes, and room temperature 12000g is centrifuged 15 minutes, are divided into two layers.Wherein supernatant liquid connects down
Carry out the extraction for RNA, lower liquid is subsequently used for the extraction of DNA.The density of pure phenol is 1.07g/cm3, mixed with water
When positioned at water phase lower layer.Chloroform becomes apparent from the layering of two kinds of solvents, because chloroform dissolves each other with phenol, and has bigger
Density 1.47g/cm3。
(2) RNA is extracted:
(1) carefully supernatant liquid is taken out, isopropanol is added dropwise, room temperature 100rpm slowly shakes after ten minutes, room temperature
12000g is centrifuged 15 minutes, discards whole liquid；Isopropanol can be mixed with the water of arbitrary proportion, therefore addition isopropanol can be taken by force
The moisture around RNA or DNA is taken, therefore RNA can assemble and precipitate, to achieve the purpose that precipitate RNA.
(2) 75% ethyl alcohol 1ml of 4 DEG C of pre-coolings is added, room temperature 12000g is centrifuged 15 minutes, blots whole liquid with vacuum pump
Body opens centrifuge tube and is placed at room temperature for 5 minutes；Its purpose is to remove remaining DNA, while solvent being made to volatilize, dry RNA.
(3) 10ul DEPC water is added, dissolves RNA.
RNA concentration, concentration 51ug/ul are measured using 2000 ultramicrospectrophotometer of Nanodrop.Through RNA fine jade
Sepharose denaturing electrophoretic analyzes visible apparent 28,18s band and thin 5s band.
(3) extraction of DNA
(1) lower liquid is carefully sucked out, 0.2ml dehydrated alcohol is added, room temperature 100rpm shakes 5 minutes, 2000g centrifugation 5
Minute, whole liquid are carefully blotted with vacuum pump；Purpose is that DNA is precipitated in order to obtain.
(2) 0.1M sodium citrate ethanol solution 0.5ml is added, room temperature 100rpm shakes after ten minutes, and 2000g is centrifuged 5 points
Clock carefully blots liquid with vacuum pump；Its purpose is to remove impurity, DNA is cleaned.
(3) 0.5ml 8mmol/L sodium hydroxide solution is added, 12000rpm is centrifuged 10 minutes, supernatant is transferred to new
In centrifuge tube, pH value is adjusted to 7.0 using HEPS, it is therefore an objective to obtain stable DNA solution.
Its concentration, concentration are measured using 2000 ultramicrospectrophotometer of Nanodrop are as follows: 52ug/ul.
Embodiment two
(1) processing of tissue sample
After taking 10mg test serum sample (mouse liver tissue) to be homogenized in liquid nitrogen, it is added as in 1 embodiment two of table
Reagent 1ml, room temperature 100rpm shake after ten minutes, and room temperature 15000g is centrifuged 10 minutes, are divided into two layers.Wherein supernatant liquid connects down
Carry out the extraction for RNA, lower liquid is subsequently used for the extraction of DNA.
(2) RNA is extracted
(1) carefully supernatant liquid is taken out, isopropanol is added dropwise, after room temperature 200rpm slowly shakes 15 minutes, room temperature
15000g is centrifuged 10 minutes, discards whole liquid.
(2) 75% ethyl alcohol 1ml of 4 DEG C of pre-coolings is added, room temperature 15000g is centrifuged 10 minutes, blots whole liquid with vacuum pump
Body opens centrifuge tube and is placed at room temperature for 5 minutes.
(3) 10ul DEPC water is added, dissolves RNA.
Its concentration, concentration 44ug/ul are measured using ultramicrospectrophotometer.Through RNA agarose gel denaturation electricity
Visible apparent 28,18s band and thin 5s band are analyzed in swimming.
(3) extraction of DNA
(1) lower liquid is carefully sucked out, 0.2ml dehydrated alcohol is added, room temperature 200rpm shakes 10 minutes, 2000g centrifugation 5
Minute, whole liquid are carefully blotted with vacuum pump.
(2) 0.05M sodium citrate ethanol solution 0.5ml is added, room temperature 200rpm shakes after five minutes, and 2000g is centrifuged 5 points
Clock carefully blots liquid with vacuum pump.
(3) be added 0.5ml 8mmol/L sodium hydroxide solution, 15000rpm be centrifuged 5 minutes, by supernatant be transferred to it is new from
In heart pipe, pH value is adjusted to 7.0 using HEPS, obtains stable DNA solution.
Ultramicrospectrophotometer is used to measure the concentration of its DNA as 38ug/ul.
Embodiment three
(1) processing of tissue sample
After taking 10mg test serum sample (mouse liver tissue) to be homogenized in liquid nitrogen, it is added as in 1 embodiment three of table
Reagent 1ml, room temperature 200rpm shake after five minutes, and room temperature 10000g is centrifuged 20 minutes, are divided into two layers.Wherein supernatant liquid connects down
Carry out the extraction for RNA, lower liquid is subsequently used for the extraction of DNA.
(2) RNA is extracted:
(1) carefully supernatant liquid is taken out, isopropanol is added dropwise, the slow 50rpm of room temperature shakes after twenty minutes, room temperature
10000g is centrifuged 20 minutes, discards whole liquid.
(2) 75% ethyl alcohol 1ml of 4 DEG C of pre-coolings is added, room temperature 10000 is centrifuged 20 minutes, whole liquid are blotted with vacuum pump,
It opens centrifuge tube and is placed at room temperature for 5 minutes.
(3) 10ul DEPC water is added, dissolves RNA.
Its concentration, concentration 33ug/ul are measured using ultramicrospectrophotometer.Through RNA agarose gel denaturation electricity
Visible apparent 28,18s band and thin 5s band are analyzed in swimming.
(3) extraction of DNA
(1) lower liquid is carefully sucked out, 0.2ml dehydrated alcohol is added, room temperature 50rpm shakes 10 minutes, 2000g centrifugation 5
Minute, whole liquid are carefully blotted with vacuum pump.
(2) 0.2M sodium citrate ethanol solution 0.5ml is added, room temperature 50rpm concussion is placed after ten minutes, 2000g centrifugation 5
Minute, liquid is carefully blotted with vacuum pump.
(3) 0.5ml 8mmol/L sodium hydroxide solution is added, 10000rpm is centrifuged 20 minutes, supernatant is transferred to new
In centrifuge tube, pH value is adjusted to 7.0 using HEPS, obtains stable DNA solution.
Ultramicrospectrophotometer is used to measure the concentration of its DNA as 35ug/ul.
Example IV
After taking 10mg test serum sample (mouse liver tissue) to be homogenized in liquid nitrogen, it is added as in 1 example IV of table
Reagent 1ml, then according to the extraction for carrying out DNA and RNA the step of embodiment one.RNA is measured using ultramicrospectrophotometer
Concentration, concentration are as follows: 25ug/ul, visible apparent 28, the 18s band and thin through RNA agarose gel denaturation electrophoretic analysis
5s band.The concentration for measuring DNA is 27ug/ul.
Embodiment five
After taking 10mg test serum sample (mouse liver tissue) to be homogenized in liquid nitrogen, it is added as in 1 embodiment five of table
Reagent 1ml, then according to the extraction for carrying out DNA and RNA the step of embodiment one.RNA is measured using ultramicrospectrophotometer
Concentration, concentration are as follows: 28ug/ul, visible apparent 28, the 18s band and thin through RNA agarose gel denaturation electrophoretic analysis
5s band, the concentration for measuring DNA is 22ug/ul.
Embodiment six
After taking 10mg test serum sample (mouse liver tissue) to be homogenized in liquid nitrogen, it is added as in 1 embodiment six of table
Reagent 1ml, then according to the extraction for carrying out DNA and RNA the step of embodiment one.RNA is measured using ultramicrospectrophotometer
Concentration, concentration are as follows: 24ug/ul, visible apparent 28, the 18s band and thin through RNA agarose gel denaturation electrophoretic analysis
5s band, the concentration for measuring DNA is 23ug/ul.
Comparative example one
After taking 10mg test serum sample (mouse liver tissue) to be homogenized in liquid nitrogen, it is added as in 1 comparative example one of table
Reagent 1ml, then according to the extraction for carrying out DNA and RNA the step of embodiment one.
Using the concentration of ultramicrospectrophotometer measurement RNA, concentration are as follows: 8ug/ul, through RNA agarose gel denaturation
Electrophoretic analysis has no obvious RNA band, shows that RNA degradation is serious, and the concentration for measuring DNA is 5ug/ul.
Comparative example two
After taking 10mg test serum sample (mouse liver tissue) to be homogenized in liquid nitrogen, it is added as in 1 comparative example two of table
Reagent 1ml, then according to the extraction for carrying out DNA and RNA the step of embodiment one.It is measured using ultramicrospectrophotometer
The concentration of RNA, concentration are as follows: 4ug/ul has no obvious RNA band, show RNA through RNA agarose gel denaturation electrophoretic analysis
Degradation is serious, and the concentration for measuring DNA is 3ug/ul.
Comparative example three
After taking 10mg test serum sample (mouse liver tissue) to be homogenized in liquid nitrogen, it is added as in 1 comparative example three of table
Reagent 1ml, then according to the extraction for carrying out DNA and RNA the step of embodiment one.It is measured using ultramicrospectrophotometer
The concentration of RNA, concentration are as follows: 3ug/ul has no obvious RNA band, show RNA through RNA agarose gel denaturation electrophoretic analysis
Degradation is serious, and the concentration for measuring DNA is 2ug/ul.
Comparative example four
After taking 10mg test serum sample (mouse liver tissue) to be homogenized in liquid nitrogen, it is added as in 1 comparative example four of table
Reagent 1ml, then according to the extraction for carrying out DNA and RNA the step of embodiment one.It is measured using ultramicrospectrophotometer
The concentration of RNA, concentration 3ug/ul have no obvious RNA band, show RNA through RNA agarose gel denaturation electrophoretic analysis
Degradation is serious, and the concentration for measuring DNA is 2ug/ul.
Comparative example five
After taking 10mg test serum sample (mouse liver tissue) to be homogenized in liquid nitrogen, it is added as in 1 comparative example five of table
Reagent 1ml, then according to the extraction for carrying out DNA and RNA the step of embodiment one.It is measured using ultramicrospectrophotometer
The concentration of RNA, concentration are as follows: 1ug/ul has no obvious RNA band, show RNA through RNA agarose gel denaturation electrophoretic analysis
Degradation is serious, and the concentration for measuring DNA is 2ug/ul.
In short, passing through the corresponding data in the above comparative example and embodiment, it is seen that, the present invention mentions RNA and DNA
It takes reagent to be optimized, strengthens the protection of DNA and RNA, room temperature in entire extraction process, without carrying out at cooling
Reason, and the needs of the subsequent sequencing of two generations and three generations's sequencing can be fully met.Operating method is simple and fast, sample requirements
Few, DNA the and RNA purity and concentration of extraction are high.
The foregoing is merely present pre-ferred embodiments, are not used to the limitation present invention, all in spirit and original of the invention
The modifications, equivalent substitutions and improvements etc. done within then are required within the protection scope of invention.
Claims (26)
1. a kind of reagent extracted simultaneously for DNA in sample and RNA, which is characterized in that
The reagent by natural saccharide compound, heavy metal salt compounds, neutral salt compounds, thiocyanic acid class compound,
Alcohol compound, organic solvent class compound, phenolic compound, N- dodecane acyl group sarcosine and for adjusting solution acid alkalinity
Salt compounds composition；
Wherein, the concentration of each compound is respectively as follows: in the reagent
(1) natural saccharide compound is 0.1-0.4mol/L；
(2) heavy metal salt compounds are 0.05-0.5mol/L；
(3) neutral salt compounds 1.0-2.0mol/L；
(4) thiocyanic acid class compound 0.9-3.0mol/L；
(5) alcohol compound 4-15% (v/v)；
(6) organic solvent class compound 1.55-7.5mol/L；
(7) phenolic compound 35%-65% (v/v)；
(8) N- dodecane acyl group sarcosine 0.01-0.07mol/L；
(9) for adjusting the salt compounds 0.8-2.5mol/L of solution acid alkalinity；
The natural saccharide compound is selected from trehalose (α, α type), isotrehalose (β, β type), one in neotrehalose (α, β type)
Kind or more；
The heavy metal salt compounds are selected from one of silver nitrate, copper sulphate, frerrous chloride, zinc chloride or more；
The neutrality salt compounds are selected from one of sodium chloride, potassium chloride, magnesium chloride, calcium chloride or more；
The organic solvent class compound is selected from one of chloroform, acetone, methanol, ethyl alcohol or more；
The alcohol compound is selected from one of glycerol, normal propyl alcohol, isopropanol, n-butanol, ethylene glycol or more；
The thiocyanic acid class compound is selected from one of guanidine thiocyanate, ammonium thiocyanate or more；
The phenolic compound is selected from one of phenol, p-cresol, o-cresol, m-cresol, phloroglucin or more；
The salt compounds for adjusting solution acid alkalinity are selected from one of sodium acetate, sodium citrate or more.
2. reagent according to claim 1, which is characterized in that the concentration of each compound is respectively as follows: in the reagent
(1) natural saccharide compound is 0.2-0.3mol/L；
(2) heavy metal salt compounds are 0.1-0.2mol/L；
(3) neutral salt compounds are 1.0-1.4mol/L；
(4) thiocyanic acid class compound is 0.9-1.5mol/L；
(5) alcohol compound is 4-6% (v/v)；
(6) organic solvent class compound is 1.55-3.1mol/L；
(7) phenolic compound is 35%-45% (v/v)；
(8) N- dodecane acyl group sarcosine is 0.03-0.06mol/L；
(9) salt compounds for adjusting solution acid alkalinity are 0.8-1.2mol/L.
3. reagent according to claim 1 or 2, which is characterized in that the natural saccharide compound is selected from trehalose (α, α
Type), isotrehalose (β, β type), one of neotrehalose (α, β type) or more；
The heavy metal salt compounds are copper sulphate or silver nitrate；
The neutrality salt compounds are sodium chloride or potassium chloride；
The organic solvent class compound is the composition of chloroform and ethyl alcohol；
The alcohol compound is glycerol or normal propyl alcohol；
The thiocyanic acid class compound is the composition of guanidine thiocyanate and ammonium thiocyanate；
The phenolic compound is phenol；
The salt compounds for adjusting solution acid alkalinity are the composition of sodium citrate and sodium acetate.
4. reagent according to claim 1, which is characterized in that the reagent is by trehalose (α, α type), copper sulphate, chlorination
Sodium, guanidine thiocyanate, ammonium thiocyanate, ethyl alcohol, glycerol, chloroform, phenol, N- dodecane acyl group sarcosine, sodium citrate and sodium acetate
Composition, wherein the concentration of each component is respectively as follows: trehalose (α, α type) 0.1-0.4mol/L in the reagent；Copper sulphate 0.05-
0.5mol/L；Sodium chloride 1.0-2.0mol/L；Guanidine thiocyanate 0.6-2.0mol/L；Ammonium thiocyanate 0.3-1.0mol/L；Glycerol
4%-15% (v/v)；Chloroform 1.5-5.0mol/L；Ethyl alcohol 0.05-2.5mol/L；Phenol 35%-65% (v/v)；N- dodecane
Acyl group sarcosine 0.01-0.07mol/L；Sodium citrate 0.7-1.5mol/L；Sodium acetate 0.1-1.0mol/L.
5. reagent according to claim 4, which is characterized in that the reagent is by trehalose (α, α type), copper sulphate, chlorination
Sodium, guanidine thiocyanate, ammonium thiocyanate, ethyl alcohol, glycerol, chloroform, phenol, N- dodecane acyl group sarcosine, sodium citrate and sodium acetate
Composition, wherein it is 0.2-0.3mol/L that the concentration of each component, which is respectively as follows: trehalose (α, α type), in the reagent；Copper sulphate 0.1-
0.2mol/L；Sodium chloride is 1.0-1.4mol/L；Guanidine thiocyanate is 0.6-1.0mol/L；Ammonium thiocyanate is 0.3-0.5mol/L；
Glycerol is 4-6% (v/v)；Chloroform is 1.5-2.0mol/L；Ethyl alcohol is 0.05-1.1mol/L；Phenol 35-45% (v/v)；N- ten
Dialkanoyl sarcosine is 0.03-0.06mol/L；Sodium citrate is 0.7-1.0mol/L；Sodium acetate is 0.1-0.2mol/L.
6. the method that the described in any item reagents of claim 1-5 are extracted for DNA and RNA, which is characterized in that including walking as follows
It is rapid:
(1) the described in any item reagents of claim 1-5 are added in sample, are centrifuged after concussion, are divided into supernatant liquid and lower layer
Liquid；
(2) wherein, the extraction of RNA is used in supernatant liquid；
(3) lower liquid is used for the extraction of DNA.
7. according to the method described in claim 6, it is characterized in that, the concussion time is 5-10min, centrifugal speed in step (1)
For 10000g-15000g, centrifugation time is 10-20 minutes.
8. method according to claim 6 or 7, which is characterized in that isopropanol rear chamber is added in liquid at the middle and upper levels for step (2)
Temperature concussion, centrifugation discard liquid；
Then 75% ethyl alcohol is added, room temperature centrifugation removes liquid, obtains RNA.
9. according to the method described in claim 8, concussion speed is it is characterized in that, shaking 10-20 minutes after isopropanol is added
50rpm-200rpm；Centrifugal speed 10000g-15000g, centrifugation time are 10-20 minutes.
10. according to the method described in claim 8, it is characterized in that, centrifugal speed is after step (2) 75% ethyl alcohol of addition
10000g-15000g, centrifugation time are 10-20 minutes.
11. method according to claim 6 or 7, which is characterized in that dehydrated alcohol is added in lower liquid in step (3)
Room temperature concussion centrifugation afterwards, removes liquid；Then sodium citrate ethanol solution is added, removes liquid after room temperature concussion centrifugation, obtains
DNA。
12. according to the method described in claim 8, it is characterized in that, dehydrated alcohol rear chamber is added in step (3) in lower liquid
Temperature concussion centrifugation, removes liquid；Then sodium citrate ethanol solution is added, removes liquid after room temperature concussion centrifugation, obtains DNA.
13. according to the method described in claim 9, it is characterized in that, dehydrated alcohol rear chamber is added in step (3) in lower liquid
Temperature concussion centrifugation, removes liquid；Then sodium citrate ethanol solution is added, removes liquid after room temperature concussion centrifugation, obtains DNA.
14. according to the method for claim 11, which is characterized in that after dehydrated alcohol is added in step (3), the concussion time is
5-10min, concussion speed are 50rpm-200rpm；
After sodium citrate ethanol solution is added, the concussion time is 5-10 minutes, and concussion speed is 50rpm-200rpm.
15. according to the method for claim 12, which is characterized in that after dehydrated alcohol is added in step (3), the concussion time is
5-10min, concussion speed are 50rpm-200rpm；
After sodium citrate ethanol solution is added, the concussion time is 5-10 minutes, and concussion speed is 50rpm-200rpm.
16. according to the method for claim 13, which is characterized in that after dehydrated alcohol is added in step (3), the concussion time is
5-10min, concussion speed are 50rpm-200rpm；
After sodium citrate ethanol solution is added, the concussion time is 5-10 minutes, and concussion speed is 50rpm-200rpm.
17. according to the method for claim 11, which is characterized in that sodium citrate in the sodium citrate ethanol solution
Concentration is 0.05M-0.2M.
18. according to the method for claim 12, which is characterized in that sodium citrate in the sodium citrate ethanol solution
Concentration is 0.05M-0.2M.
19. according to the method for claim 13, which is characterized in that sodium citrate in the sodium citrate ethanol solution
Concentration is 0.05M-0.2M.
20. according to the method for claim 14, which is characterized in that sodium citrate in the sodium citrate ethanol solution
Concentration is 0.05M-0.2M.
21. according to the method for claim 15, which is characterized in that sodium citrate in the sodium citrate ethanol solution
Concentration is 0.05M-0.2M.
22. according to the method for claim 16, which is characterized in that sodium citrate in the sodium citrate ethanol solution
Concentration is 0.05M-0.2M.
23. the described in any item reagents of claim 1-5 or the described in any item methods of claim 6-22 for extracting and/or
Measure the application in the content of the DNA and RNA in sample.
24. application according to claim 23, the DNA and RNA of the application fetches can be used as genome sequencing, exon
Sequencing, specific region sequencing.
25. application according to claim 23, the sample in the application includes fresh pathological tissues, tumor tissues disease
Example wax stone, tumor tissues case paraffin section, whole blood, blood plasma, serum or pleural effusion.
26. application according to claim 23, the sample in the application includes fresh biopsy sample.
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| CN110904094B (en) * | 2019-12-10 | 2021-07-30 | 北京市理化分析测试中心 | Extraction method of salivary plaque miRNA and method for constructing salivary plaque miRNA high-throughput sequencing library |
| CN113462682A (en) * | 2021-06-18 | 2021-10-01 | 汉远化生医国际科技(北京)有限公司 | Lysis, binding, washing and/or elution reagents for the isolation and/or purification of biological RNA |
| CN114908081A (en) * | 2022-04-29 | 2022-08-16 | 杭州新景生物试剂开发有限公司 | Nucleic acid extraction reagent, kit thereof and method for rapidly extracting, separating and purifying nucleic acid |
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| CN100439509C (en) * | 2005-06-28 | 2008-12-03 | 国家***第三海洋研究所 | Method of simultaneously extracting microorganism macrogenome DNA and total DNA from sea precipitate |
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