CN106906207A - Rapid nucleic acid extraction kit and its application method - Google Patents
Rapid nucleic acid extraction kit and its application method Download PDFInfo
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- CN106906207A CN106906207A CN201710258358.8A CN201710258358A CN106906207A CN 106906207 A CN106906207 A CN 106906207A CN 201710258358 A CN201710258358 A CN 201710258358A CN 106906207 A CN106906207 A CN 106906207A
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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
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Abstract
This application discloses Rapid nucleic acid extraction kit, include mixed enzyme working solution, cracking adsorption liquid, cleaning solution, rinsing liquid and the eluent individually prepared in the kit, the mixed enzyme working solution includes Proteinase K and lysozyme, the cracking adsorption liquid includes Gu Hcl, Tris Hcl, EDTA, isopropanol, SDS, Triton 100, β mercaptoethanols and sodium citrate, the cleaning solution includes Licl, Nacl, MOPS and ethanol, the rinsing liquid is ethanol, and the eluent is EDTA and Tris Hcl.Kit of the invention can be applicable whole blood, cultured cells, tissue, saliva, G‑Bacterium, G+The extraction of the genomic DNAs such as bacterium, extract gained DNA purity high, be applicable to digestion and the PCR biological operations in downstream, agents useful for same is stable in properties, suitable for the demand of common laboratory, while the shortcomings of also overcoming that conventional method is poisonous, time-consuming and be cumbersome.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to based on paramagnetic particle method from whole blood, cultured cells, tissue,
Rapid nucleic acid extraction kit and its extraction side of genomic DNA are extracted in the common clinical samples such as saliva, G- bacteriums, G+ bacteriums
Method.
Background technology
The isolation and purification technology of nucleic acid is a basic fundamental of molecular biology experiment, is carried from initial genes of interest
The experimentations such as the extraction of plasmid in PCR primer purifying or " glue reclaim " and gene cloning are got, nucleic acid extraction is all be unable to do without
Purifying.Nucleic acid separating and purifying technology originates from the 1950's, traditional nucleic acid precipitation dissolving in the seventies and the eighties
Isolation and purification method has obtained universal application and popularization.Conventional method for extracting nucleic acid is related to cell to crack, organic solvent extracts
The step such as take, precipitate and be centrifuged.This process wastes time and energy, and is difficult automation, and with the quick hair of biological and medical technology
Exhibition, the method for original manual purification of nucleic acid has been difficult to meet extensive, high-throughout requirement of experiment.
The content of the invention
The present invention is difficult to meet the problem of extensive, high-throughout requirement of experiment for prior art nucleic acid extraction, improves
A kind of simple and rapid Magnetic Isolation kit based on magnetic separation technique.
Concrete scheme is：Rapid nucleic acid extraction kit, the interior mixed enzyme working solution for including individually preparing of the kit,
Cracking adsorption liquid, cleaning solution, rinsing liquid and eluent, the mixed enzyme working solution include Proteinase K and lysozyme, the cracking
Adsorption liquid includes Gu-Hcl, Tris-Hcl, EDTA, isopropanol, SDS, Triton-100, beta -mercaptoethanol and sodium citrate, institute
Stating cleaning solution includes Licl, Nacl, MOPS and ethanol, and the rinsing liquid is ethanol, and the eluent is EDTA and Tris-Hcl.
Wherein, Gu-Hcl is guanidine hydrochloride, and the alias of Tris-Hcl is three (methylol) aminomethanes, tromethamine, slow blood
Sour ammonium or trishydroxymethylaminomethane, molecular formula are C4H11NO3；EDTA is ethylenediamine tetra-acetic acid, and molecular formula is C10H16N2O8；
SDS is lauryl sodium sulfate；The alias that Triton-100 is is Triton X-100 or polyoxethylene octylphenyl benzene
Base ether, Licl is lithium chloride, and MOPS is 3- (N- morpholines) propane sulfonic acid.
Further, in the mixed enzyme working solution, the concentration of Proteinase K is 10 ~ 40mg/mL, and the concentration of lysozyme is
10~40mg/mL。
Further, in the cracking adsorption liquid, the concentration of Gu-Hcl is 3 ~ 8mol/L, the concentration of Tris-Hcl for 10 ~
The concentration of 30mmol/L, EDTA is the volume ratio of 1 ~ 10mmol/L, isopropanol for 1/2 ~ 1/6, SDS is 0.1 ~ 0.5%, Triton-
100 is 0.5 ~ 2%, and beta -mercaptoethanol is 800mmol/L for the concentration of 0.5 ~ 2%, Nacl, and the concentration of sodium citrate is 200mmol/
The pH=6.8 of L, wherein sodium citrate.
Further, in the cleaning solution, the concentration of Licl is 800mmol/L, and the concentration of Nacl is 100mmol/L,
The concentration of MOPS is 50mmol/L, and the volume ratio of ethanol is 60%, the wherein pH=7.0 of ethanol.
Further, in the rinsing liquid, the concentration of ethanol is 70%.
Further, in the eluent, the concentration of EDTA is 0.5mmol/L, the concentration 10mmol/L of Tris-Hcl, its
The pH=8.0 of middle Tris-Hcl.
The application method of Rapid nucleic acid extraction kit, comprises the following steps：Step one, 100 μ L samples are taken with test tube, plus
Enter 10 μ L mixed enzyme working solutions, 56 DEG C of water-bath 5min；
Step 2, addition 750 μ L cracking adsorption liquid and magnetic Nano microsphere, the test tube that turns upside down is sufficiently mixed for 5-10 times, in room
Temperature is lower to place 3min；
Step 3, with Magneto separate frame separate magnetic bead abandon supernatant；
Step 4, washed 2 times with 800 μ L cleaning solutions, separate magnetic bead and abandon supernatant；
Step 5, with 1000 μ L rinsing liquids rinse 2 times afterwards separate magnetic bead abandon supernatant；
Step 6, room temperature place 3min, volatilize ethanol, add 100 μ L eluents to blow and beat precipitation up and down, microballoon is fully suspended,
3min is placed at room temperature, obtains the DNA of purifying.
Described sample is whole blood, cultured cells, tissue, saliva, G- bacteriums and G+ bacteriums.
Paramagnetic particle method nucleic acid with nanometer technology the surface of superparamagnetic nano particle is improved and surface modification after, system
For into superparamagnetism silica nanometer magnetic bead, the magnetic bead can specifically be recognized and efficiently tied on micro interface with nucleic acid molecules
Close, using the superparamagnetism of silica Nano microsphere, in Chaotropic salt（Guanidine hydrochloride, guanidinium isothiocyanate etc.）And externally-applied magnetic field
In the presence of, can be separated from DNA and RNA in blood, animal tissue, food, pathogenic microorganism equal samples, can be applicable to
Clinical disease diagnosis, transfusion safety, Forensic Identification, environmental microorganism detection, food safety detection, molecular biology research etc.
Multiple fields.
Extracts kit of the invention and experimental technique have the following advantages that：1st, it is cheap, go for various samples
This is such as：Whole blood, cultured cells, tissue, saliva, G-Bacterium, G+The extraction of the genomic DNAs such as bacterium；2nd, body series extract gained
DNA purity is high, is applicable to digestion and the PCR biological operations in downstream；3rd, body series do not need centrifugally operated, special experiment bar
The instrument and equipment and easy to automate of part and costliness；4th, body series agents useful for same is stable in properties, it is adaptable to common laboratory
Demand, while also overcome that conventional method is poisonous, time-consuming, it is cumbersome the shortcomings of.
Brief description of the drawings
Fig. 1 is the electrophoresis detection figure of the Whole Blood Genomic DNA extracted using kit of the present invention and similar kit.
Specific embodiment
Below by specific embodiment, the present invention is further detailed explanation：
Specific embodiment of the invention, but protection scope of the present invention are illustrated by example below, this is not limited to.
Embodiment 1：By taking whole blood sample as an example：
Step one, 100 μ L anticoagulations are taken, add 10 μ L mixed enzyme working solutions, 56 DEG C of water-bath 5min.
Step 2, turn upside down test tube 5-10 time of 750 μ L cracking adsorption liquids and appropriate magnetic Nano microsphere is added fully to mix
Room temperature places 3min after conjunction.
Step 3, with Magneto separate frame separate magnetic bead abandon supernatant.
Step 4, washed 2 times with 800 μ L cleaning solutions, separate magnetic bead and abandon supernatant.
Step 5, with 1000 μ L rinsing liquids rinse 2 times afterwards separate magnetic bead abandon supernatant.
Step 6, room temperature place 3min, volatilize ethanol, and adding 100 μ L eluents to blow and beat precipitation up and down makes microballoon fully hang
Placed at room temperature 3 minutes after floating, obtain the DNA of purifying.
Wherein, the concentration of Proteinase K is 10mg/mL, and the concentration of lysozyme is 10mg/mL.
In cracking adsorption liquid, the concentration of Gu-Hcl is 3mol/L, and the concentration of Tris-Hcl is 10mmol/L, the concentration of EDTA
For 1mmol/L, isopropanol volume ratio for 1/2, SDS be that 0.1%, Triton-100 is 0.5%, beta -mercaptoethanol is 0.5%,
The concentration of Nacl is 800mmol/L, and the concentration of sodium citrate is 200mmol/L, the wherein pH=6.8 of sodium citrate.
In cleaning solution, the concentration of Licl is 800mmol/L, and the concentration of Nacl is 100mmol/L, and the concentration of MOPS is
50mmol/L, the volume ratio of ethanol is 60%, the wherein pH=7.0 of ethanol.
In eluent, the concentration of EDTA is 0.5mmol/L, the concentration 10mmol/L of Tris-Hcl, wherein Tris-Hcl's
pH=8.0。
Embodiment 2 is 20mg/mL with the concentration for differing only in Proteinase K of embodiment 1, and the concentration of lysozyme is
20mg/mL。
In cracking adsorption liquid, the concentration of Gu-Hcl is 4.4mol/L, and the concentration of Tris-Hcl is 10mmol/L, and EDTA's is dense
The volume ratio for 1mmol/L, isopropanol is spent for 1/4, SDS is that 0.1%, Triton-100 is 1%, and beta -mercaptoethanol is 1%.
Embodiment 3 is 40mg/mL with the concentration for differing only in Proteinase K of embodiment 1, and the concentration of lysozyme is
40mg/mL。
In cracking adsorption liquid, the concentration of Gu-Hcl is 8mol/L, and the concentration of Tris-Hcl is 30mmol/L, the concentration of EDTA
For 10mmol/L, isopropanol volume ratio for 1/6, SDS be that 0.5%, Triton-100 is 2%, beta -mercaptoethanol is 2%.
Embodiment 4：By taking saliva sample as an example：
Step one, the μ L of the saliva sample 100 plus μ L of physiological saline 100 are taken, fully mixed, diluted standby.
Step 2, take dilution after the μ L of sample liquid 100, add 10 μ L mixed enzyme working solutions, 56 DEG C of water-bath 5min.
Step 3, turn upside down test tube 5-10 time of 750 μ L cracking adsorption liquids and appropriate magnetic Nano microsphere is added fully to mix
Room temperature places 3min after conjunction.
Step 4, with Magneto separate frame separate magnetic bead abandon supernatant.
Step 5, washed 2 times with 800 μ L cleaning solutions, separate magnetic bead and abandon supernatant.
Step 6, with 1000 μ L rinsing liquids rinse 2 times afterwards separate magnetic bead abandon supernatant.
Step 7, room temperature place 3min, volatilize ethanol, and adding 100 μ L eluents to blow and beat precipitation up and down makes microballoon fully hang
Placed at room temperature 3 minutes after floating, obtain the DNA of purifying.
With embodiment 2, similar kit, Takara DL2000 DNA Marker and physiological saline are contrast experiment, such as
Shown in Fig. 1, Fig. 1 is the electrophoresis detection figure of the Whole Blood Genomic DNA extracted using kit of the present invention and similar kit.
A total of 11 sample holes, reagent to be detected is injected using 1 ~ 6 sample hole, and sample hole 1 is DNA sample-loading buffers（6×
quadricolor-loading buffer）The blank for doing, is whole blood base that kit of the present invention is extracted in sample hole 2,3
It is the Whole Blood Genomic DNA sample that similar kit is extracted because of a group DNA sample, in sample hole 4,5, sample hole 6 is Takara
DL2000 DNA Marker samples, sample hole correspondence swimming lane.As shown in figure 1, swimming lane 1 is blank testing result, swimming lane 2,3
It is the Whole Blood Genomic DNA testing result that kit of the present invention is extracted, swimming lane 4,5 is the whole blood base that similar kit is extracted
Because a group DNA testing results, swimming lane 6 are Takara DL2000 DNA Marker, the DNA band brightness of 2,3 swimming lanes is significantly stronger than
4th, 5 swimming lane, illustrates on the premise of identical applied sample amount, and the concentration that this kit extracts gained DNA sample is substantially better than similar product
Product.
Experimental result comparative analysis：
In terms of DNA mass, the genome DNA sample purity that this kit is extracted is high, and protein content is low, and salt residual is few, can be straight
Connect for PCR amplifications；DNA extract timeliness in terms of, this kit single-trial extraction time in 30min or so, better than numerous domestic
Kit.
Above-described is only embodiments of the invention, and the general knowledge such as known concrete structure and characteristic is not made herein in scheme
Excessive description.It should be pointed out that for a person skilled in the art, on the premise of structure of the present invention is not departed from, can be with
Some deformations and improvement are made, these should also be considered as protection scope of the present invention, these are implemented all without the influence present invention
Effect and practical applicability.This application claims protection domain should be defined by the content of its claim, in specification
Specific embodiment etc. records the content that can be used for explaining claim.
Claims (7)
1. Rapid nucleic acid extraction kit, it is characterised in that include the mixed enzyme working solution individually prepared in the kit, split
Desorption attached liquid, cleaning solution, rinsing liquid and eluent, the mixed enzyme working solution include Proteinase K and lysozyme, and the cracking is inhaled
Attached liquid includes Gu-Hcl, Tris-Hcl, EDTA, isopropanol, SDS, Triton-100, beta -mercaptoethanol and sodium citrate, described
Cleaning solution includes Licl, Nacl, MOPS and ethanol, and the rinsing liquid is ethanol, and the eluent is EDTA and Tris-Hcl.
2. Rapid nucleic acid extraction kit according to claim 1, it is characterised in that in the mixed enzyme working solution, egg
The concentration of white enzyme K is 10 ~ 40mg/mL, and the concentration of lysozyme is 10 ~ 40mg/mL.
3. Rapid nucleic acid extraction kit according to claim 1, it is characterised in that in the cracking adsorption liquid, Gu-
The concentration of Hcl is 3 ~ 8mol/L, and the concentration of Tris-Hcl is 10 ~ 30mmol/L, and the concentration of EDTA is 1 ~ 10mmol/L, isopropanol
Volume ratio for 1/2 ~ 1/6, SDS be 0.1 ~ 0.5%, Triton-100 is 0.5 ~ 2%, beta -mercaptoethanol is for 0.5 ~ 2%, Nacl
Concentration is 800mmol/L, and the concentration of sodium citrate is 200mmol/L, the wherein pH=6.8 of sodium citrate.
4. Rapid nucleic acid extraction kit according to claim 1, it is characterised in that in the cleaning solution, Licl's is dense
It is 800mmol/L to spend, and the concentration of Nacl is 100mmol/L, and the concentration of MOPS is 50mmol/L, and the volume ratio of ethanol is 60%, its
The pH=7.0 of middle ethanol.
5. Rapid nucleic acid extraction kit according to claim 1, it is characterised in that in the rinsing liquid, ethanol it is dense
Spend is 70%.
6. Rapid nucleic acid extraction kit according to claim 1, it is characterised in that in the eluent, EDTA's is dense
It is 0.5mmol/L to spend, the concentration 10mmol/L of Tris-Hcl, the wherein pH=8.0 of Tris-Hcl.
7. according to the application method of any described Rapid nucleic acid extraction kit of claim 1 ~ 6, it is characterised in that including with
Lower step：Step one, 100 μ L samples are taken with test tube, add 10 μ L mixed enzyme working solutions, 56 DEG C of water-bath 5min；
Step 2, addition 750 μ L cracking adsorption liquid and magnetic Nano microsphere, the test tube that turns upside down is sufficiently mixed for 5 ~ 10 times, in room
Temperature is lower to place 3min；
Step 3, with Magneto separate frame separate magnetic bead abandon supernatant；
Step 4, washed 2 times with 800 μ L cleaning solutions, separate magnetic bead and abandon supernatant；
Step 5, with 1000 μ L rinsing liquids rinse 2 times afterwards separate magnetic bead abandon supernatant；
Step 6, room temperature place 3min, volatilize ethanol, add 100 μ L eluents to blow and beat precipitation up and down, microballoon is fully suspended,
3min is placed at room temperature, obtains the DNA of purifying.
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| CN107326024A (en) * | 2017-09-07 | 2017-11-07 | 辽宁省农业科学院大连生物技术研究所 | A kind of high flux rapid extraction plasmodium DNA method and its application |
| CN108866044A (en) * | 2018-07-23 | 2018-11-23 | 华中科技大学鄂州工业技术研究院 | A kind of method of rapidly extracting genomic DNA |
| CN109055354A (en) * | 2018-07-23 | 2018-12-21 | 华中科技大学鄂州工业技术研究院 | A kind of genomic DNA rapidly extracting kit and preparation method thereof |
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| CN111304291A (en) * | 2020-03-30 | 2020-06-19 | 北京泛生子基因科技有限公司 | DNA extraction reagent, kit and extraction method |
| CN111607590A (en) * | 2020-06-08 | 2020-09-01 | 申翌生物科技(杭州)有限公司 | Nucleic acid extraction method and kit applicable to whole blood, serum or plasma by virtue of magnetic bead method |
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| CN113215151A (en) * | 2021-06-29 | 2021-08-06 | 杭州圣庭医疗科技有限公司 | Method for extracting DNA nucleic acid of bacteria, fungi and viruses |
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