CN110172457A - A kind of whole blood DNA extracts kit can be used for building library - Google Patents
A kind of whole blood DNA extracts kit can be used for building library Download PDFInfo
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- CN110172457A CN110172457A CN201910515904.0A CN201910515904A CN110172457A CN 110172457 A CN110172457 A CN 110172457A CN 201910515904 A CN201910515904 A CN 201910515904A CN 110172457 A CN110172457 A CN 110172457A
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- whole blood
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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
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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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The present invention relates to a kind of whole blood DNA extracts kits that can be used for building library, belong to bioengineering field, and wherein whole blood DNA extracts kit mainly includes lysate, nano biological magnetic bead, cleaning solution A, cleaning solution S and eluent；Kit of the present invention specific aim in cleaning solution A is added to the ingredient of removal inorganic salts, has the characteristics that operating procedure is simple, DNA yield is high, integrity degree is good, with high purity.Especially with the operating method that kit of the present invention and kit provide, the DNA sample that can be directly used for the sequencing of two generations simple and quick can be obtained, the construction of DNA sample storehouse is particularly suitable for.
Description
Technical field
The invention belongs to bioengineering fields, and in particular, to a kind of whole blood DNA extracts kit that can be used for building library.
Background technique
With the development of molecular biology technology, the research in genomic level has become hot spot.In molecular genetics and
In molecular epidemiology, either whole-genome association (GWAS), candidate SNP locus Genotyping or genome are literary
The foundation in library requires to extract genomic DNA from Different categories of samples.Since blood sample is drawn materials conveniently, contained genomic DNA is rich
It is rich reliable, therefore above-mentioned much researchs are using blood as the complete genomic DNA of sample extraction.The gene that extraction purification obtains
The integrality of group DNA concentration, purity and primary structure can all influence subsequent research, therefore efficient, reliable genomic DNA
Extracting method be molecular genetics basic research there is an urgent need to.
At present extracting genome DNA purifying method there are many kinds of, as phenol chloroform extraction method, SDS method, centrifugal column method and
Paramagnetic particle method.There is also a variety of whole blood DNA extracts kits in the market, but all have the disadvantage in that go to the maximum extent
Removing protein, pigment, lipid and other mortifiers；The organic solvents such as phenol, chloroform are used in extraction process；Blood sample is risen
Initial body product is restricted, influences adaptability；The DNA poor quality of extraction is unsuitable for building library.
Summary of the invention
In order to solve deficiency in the prior art, the purpose of the present invention is to provide a kind of whole blood DNAs that can be used for building library
Extracts kit, using the kit, operating procedure is simple, simple and quick can obtain can be directly used for the sequencing of two generations
DNA sample, and DNA yield is high, integrity degree is good, with high purity, is particularly suitable for the construction of DNA sample storehouse.
To achieve the goals above, the present invention use the specific scheme is that
A kind of whole blood DNA extracts kit can be used for building library, it is characterised in that include lysate, nano biological magnetic bead, washing
Liquid A, cleaning solution S and eluent；It include guanidinium isothiocyanate, guanidine hydrochloride, trisodium citrate and Tween20 in the lysate；Institute
Stating nano biological magnetic bead is the iron oxide for being enclosed with silica, polyvinyl alcohol, polyethylene glycol or cellulose；The cleaning solution A
In include cetyl trimethylammonium bromide, Tris-Hcl, dehydrated alcohol and FMES；It include Tris-Hcl in the cleaning solution S
And dehydrated alcohol；It include Tris-Hcl and EDETATE SODIUM in the eluent.
It is advanced optimized as to above scheme, the pH value of the lysate is 5.0~7.5；It is different in the lysate
The concentration of guanidine thiocyanate is 1~3mM, the concentration of guanidine hydrochloride is 2~4M, the concentration of trisodium citrate be 100~200mM,
The concentration of Tween20 is 2%~4%.
It is advanced optimized as to above scheme, the diameter of the nano biological magnetic bead is 200~3000nm, and concentration is
50~100mg/ml.
It is advanced optimized as to above scheme, the concentration of cetyl trimethylammonium bromide is in the cleaning solution A
The concentration for the Tris-Hcl that 50~100mM, pH value are 7.0~8.5 is 10~30mM, the percentage by volume of dehydrated alcohol is 30%
~70%, the concentration of FMES is 10~50mM.
It is advanced optimized as to above scheme, the Tris-Hcl's that pH value is 7.0~8.5 in the cleaning solution S is dense
Degree is 10~30mM, the percentage by volume of dehydrated alcohol is 30%~70%.
It is advanced optimized as to above scheme, the pH value of the eluent is 7.0~8.5；In the eluent
The concentration of Tris-Hcl is 10~30mM, the concentration of EDETATE SODIUM is 0.2-0.4mM's.
Advanced optimized as to above scheme, the application method of the kit the following steps are included:
Step S1: taking 50-200ul fresh whole blood sample in 1.5ml centrifuge tube, and 200-400ul is added using sterile pipette
Lysate, 20~70 DEG C of 10~30min of water-bath；
Step S2: 300~500ul isopropanol and 5~30ul nano biological magnetic bead being added into centrifuge tube using sterile pipette,
Concussion mixes 5~10min；
Step S3: being placed in 45S on magnetic frame for the centrifuge tube handled through step S2, is adsorbed to tube wall completely to nano biological magnetic bead
Later, it is inhaled using sterile pipette and abandons supernatant solution；
Step S4: 500~800ul cleaning solution A is added into step S3 centrifuge tube using sterile pipette, which is shaken
2min is mixed, 30S on magnetic frame is placed in, after being adsorbed to tube wall completely to nano biological magnetic bead, is inhaled and is abandoned using sterile pipette
Supernatant solution；
Step S5: 500~800ul cleaning solution S is added into step S4 centrifuge tube using sterile pipette, which is shaken
2min is mixed, 30S on magnetic frame is placed in, after being adsorbed to tube wall completely to nano biological magnetic bead, is inhaled and is abandoned using sterile pipette
Supernatant solution；
Step S6: being added 70~200ul eluent into step S5 centrifuge tube using sterile pipette, and 65 DEG C of water-baths 5~
Then centrifuge tube is placed in 30S on magnetic frame by 10min, after being adsorbed to tube wall completely to nano biological magnetic bead, use sterile shifting
Liquid device draws supernatant into new centrifuge tube, that is, DNA needed for obtaining.
The utility model has the advantages that
The whole blood DNA extracts kit of the present invention that can be used for building library, it is inorganic to be added to removal for specific aim in cleaning solution A
The ingredient of salt has the characteristics that operating procedure is simple, DNA yield is high, integrity degree is good, with high purity.It is tried especially with the present invention
The operating method that agent box and kit provide simple and quick can obtain the DNA sample that can be directly used for the sequencing of two generations, especially suitable
It is built for DNA sample storehouse.
Detailed description of the invention
Fig. 1 is the genomic DNA integrity degree of whole blood DNA extracts kit and QIAamp DNA Mini Kit of the invention
Comparison schematic diagram；Wherein first row M is DNA Marker, and for marking DNA molecular amount size, 2-4 is classified as embodiment 2,5-7 column
For contrast agents result.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described.
Embodiment 1
A kind of whole blood DNA extracts kit can be used for building library, comprising the following specific steps
Step S1: taking 50ul fresh whole blood sample in 1.5ml centrifuge tube, using sterile pipette be added 200ul lysate, 70
DEG C water-bath 30min；
Step S2: 300ul isopropanol, 10ul nano biological magnetic bead is added using sterile pipette, concussion mixes 5min；
Step S3: being placed in 45S on magnetic frame for the centrifuge tube in step S2, to nano biological magnetic bead be adsorbed to completely tube wall it
Afterwards, it is inhaled using sterile pipette and abandons supernatant solution；
Step S4: 500ul cleaning solution A is added into step S3 centrifuge tube using sterile pipette, which is shaken and is mixed
2min is placed in 30S on magnetic frame, after being adsorbed to tube wall completely to nano biological magnetic bead, is inhaled using sterile pipette and abandons supernatant
Solution；
Step S5: 500ul cleaning solution S is added into step S4 centrifuge tube using sterile pipette, which is shaken and is mixed
2min is placed in 30S on magnetic frame, after being adsorbed to tube wall completely to nano biological magnetic bead, is inhaled using sterile pipette and abandons supernatant
Solution；
Step S6: using sterile pipette that 70ul eluent is added into step S5 centrifuge tube, then 65 DEG C of water-bath 10min will
Centrifuge tube is placed in 30S on magnetic frame, after being adsorbed to tube wall completely to nano biological magnetic bead, draws supernatant using sterile pipette
Into new centrifuge tube, that is, DNA needed for obtaining.
Embodiment 2
A kind of whole blood DNA extracts kit can be used for building library, comprising the following specific steps
Step S1: taking 200ul fresh whole blood sample in 1.5ml centrifuge tube, and 300ul lysate is added using sterile pipette,
70 DEG C of water-bath 30min；
Step S2: 300ul isopropanol, 20ul nano biological magnetic bead is added using sterile pipette, concussion mixes 5min；
Step S3: being placed in 45S on magnetic frame for the centrifuge tube in step S2, to nano biological magnetic bead be adsorbed to completely tube wall it
Afterwards, it is inhaled using sterile pipette and abandons supernatant solution；
Step S4: 800ul cleaning solution A is added into step S3 centrifuge tube using sterile pipette, which is shaken and is mixed
2min is placed in 30S on magnetic frame, after being adsorbed to tube wall completely to nano biological magnetic bead, is inhaled using sterile pipette and abandons supernatant
Solution；
Step S5: 800ul cleaning solution S is added into step S4 centrifuge tube using sterile pipette, which is shaken and is mixed
2min is placed in 30S on magnetic frame, after being adsorbed to tube wall completely to nano biological magnetic bead, is inhaled using sterile pipette and abandons supernatant
Solution；
Step S6: using sterile pipette that 100ul eluent is added into step S5 centrifuge tube, then 65 DEG C of water-bath 5min will
Centrifuge tube is placed in 30S on magnetic frame, after being adsorbed to tube wall completely to nano biological magnetic bead, draws supernatant using sterile pipette
Into new centrifuge tube, that is, DNA needed for obtaining.
In the present invention, contrast agents selection QIAamp DNA Mini Kit extracts 200ul fresh whole blood sample, elution
Volume is 100ul.
Embodiment 3
A kind of whole blood DNA extracts kit can be used for building library, comprising the following specific steps
Step S1: taking 200ul fresh whole blood sample in 1.5ml centrifuge tube, and 400ul lysate is added using sterile pipette,
65 DEG C of water-bath 20min；
Step S2: 400ul isopropanol, 20ul nano biological magnetic bead is added using sterile pipette, concussion mixes 10min；
Step S3: being placed in 45S on magnetic frame for the centrifuge tube in step S2, to nano biological magnetic bead be adsorbed to completely tube wall it
Afterwards, it is inhaled using sterile pipette and abandons supernatant solution；
Step S4: 700ul cleaning solution A is added into step S3 centrifuge tube using sterile pipette, which is shaken and is mixed
2min is placed in 30S on magnetic frame, after being adsorbed to tube wall completely to nano biological magnetic bead, is inhaled using sterile pipette and abandons supernatant
Solution；
Step S5: 700ul cleaning solution S is added into step S4 centrifuge tube using sterile pipette, which is shaken and is mixed
2min is placed in 30S on magnetic frame, after being adsorbed to tube wall completely to nano biological magnetic bead, is inhaled using sterile pipette and abandons supernatant
Solution；
Step S6: being added 100ul eluent into step S5 centrifuge tube using sterile pipette, 65 DEG C of 5~10min of water-bath, so
Centrifuge tube is placed in 30S on magnetic frame afterwards, after being adsorbed to tube wall completely to nano biological magnetic bead, is drawn using sterile pipette
Supernatant is into new centrifuge tube, that is, DNA needed for obtaining.
In the present invention, it is 100ul that contrast agents, which select QIAamp DNA Mini Kit elution volume, as a result such as subordinate list 1
It is shown.
By embodiment 2, contrast agents, after the completion of extraction, the whole blood DNA solution 5ul obtained respectively carries out 1% agarose
Gel electrophoresis, as a result as shown in Fig. 1.
As shown in Figure 1, using the present invention is based on the whole blood DNA extracts kits of nano biological magnetic bead, by its experimental result
Be compared with the experimental result of QIAamp DNA Mini Kit, the results showed that, whole blood DNA integrity degree that the present invention extracts with
The whole blood DNA integrity degree that QIAamp DNA Mini Kit is obtained is consistent.
By embodiment 3, contrast agents, after the completion of extraction, the whole blood DNA solution 2.5ul obtained respectively is utilized
The NanoDrop2000 ultramicron nucleic acid-protein analyzer of ThermoFisher detects DNA concentration and purity, as a result such as
Shown in table 1.
Table 1:DNA concentration and comparison or purity table
As shown in Table 1, the concentration of the whole blood DNA that the present invention obtains and QIAamp DNA Mini Kit are in same level, but
In terms of purity, the present invention is better than contrast agents box, and especially on OD260/OD230, kit purity of the present invention is more preferable, is suitable for
The sequencing of two generations is directly carried out, is suitable for biological sample bank and builds.
It should be noted that embodiment described above is interpreted as illustrative, to be not intended to limit the present invention protection
Range, protection scope of the present invention are subject to claims.To those skilled in the art, without departing substantially from of the invention real
Under the premise of matter and range, some nonessential modifications and adaptations made to the present invention still fall within protection scope of the present invention.
Claims (7)
1. a kind of whole blood DNA extracts kit that can be used for building library, it is characterised in that: comprising lysate, nano biological magnetic bead, wash
Wash liquid A, cleaning solution S and eluent；It include guanidinium isothiocyanate, guanidine hydrochloride, trisodium citrate and Tween20 in the lysate；
The nano biological magnetic bead is the iron oxide for being enclosed with silica, polyvinyl alcohol, polyethylene glycol or cellulose；The cleaning solution
It include cetyl trimethylammonium bromide, Tris-Hcl, dehydrated alcohol and FMES in A；It include Tris-Hcl in the cleaning solution S
And dehydrated alcohol；It include Tris-Hcl and EDETATE SODIUM in the eluent.
2. a kind of whole blood DNA extracts kit that can be used for building library according to claim 1, it is characterised in that: described to split
The pH value for solving liquid is 5.0~7.5；In the lysate concentration of guanidinium isothiocyanate be 1~3mM, the concentration of guanidine hydrochloride be 2~
4M, trisodium citrate concentration be 100~200mM, the concentration of Tween20 is 2%~4%.
3. a kind of whole blood DNA extracts kit that can be used for building library according to claim 1, it is characterised in that: described to receive
The diameter of rice biomagnetic beads is 200~3000nm, and concentration is 50~100mg/ml.
4. a kind of whole blood DNA extracts kit that can be used for building library according to claim 1, it is characterised in that: described to wash
The concentration for washing cetyl trimethylammonium bromide in liquid A is 50~100mM, the concentration for the Tris-Hcl that pH value is 7.0~8.5
Percentage by volume for 10~30mM, dehydrated alcohol is 30%~70%, the concentration of FMES is 10~50mM.
5. a kind of whole blood DNA extracts kit that can be used for building library according to claim 1, it is characterised in that: described to wash
The concentration for washing the Tris-Hcl that pH value is 7.0~8.5 in liquid S is 10~30mM, the percentage by volume of dehydrated alcohol be 30%~
70%。
6. a kind of whole blood DNA extracts kit that can be used for building library according to claim 1, it is characterised in that: described to wash
The pH value of de- liquid is 7.0~8.5；The concentration of Tris-Hcl is 10~30mM in the eluent, the concentration of EDETATE SODIUM is
0.2-0.4mM's.
7. a kind of whole blood DNA extracts kit that can be used for building library according to claim 1, it is characterised in that: the examination
The application method of agent box the following steps are included:
Step S1: taking 50-200ul fresh whole blood sample in 1.5ml centrifuge tube, and 200-400ul is added using sterile pipette
Lysate, 20~70 DEG C of 10~30min of water-bath；
Step S2: 300~500ul isopropanol and 5~30ul nano biological magnetic bead being added into centrifuge tube using sterile pipette,
Concussion mixes 5~10min；
Step S3: being placed in 45S on magnetic frame for the centrifuge tube handled through step S2, is adsorbed to tube wall completely to nano biological magnetic bead
Later, it is inhaled using sterile pipette and abandons supernatant solution；
Step S4: 500~800ul cleaning solution A is added into step S3 centrifuge tube using sterile pipette, which is shaken
2min is mixed, 30s on magnetic frame is placed in, after being adsorbed to tube wall completely to nano biological magnetic bead, is inhaled and is abandoned using sterile pipette
Supernatant solution；
Step S5: 500~800ul cleaning solution S is added into step S4 centrifuge tube using sterile pipette, which is shaken
2min is mixed, 30s on magnetic frame is placed in, after being adsorbed to tube wall completely to nano biological magnetic bead, is inhaled and is abandoned using sterile pipette
Supernatant solution；
Step S6: being added 70~200ul eluent into step S5 centrifuge tube using sterile pipette, and 65 DEG C of water-baths 5~
Then centrifuge tube is placed in 30s on magnetic frame by 10min, after being adsorbed to tube wall completely to nano biological magnetic bead, use sterile shifting
Liquid device draws supernatant into new centrifuge tube, that is, DNA needed for obtaining.
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| CN201910515904.0A CN110172457A (en) | 2019-06-14 | 2019-06-14 | A kind of whole blood DNA extracts kit can be used for building library |
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| CN201910515904.0A CN110172457A (en) | 2019-06-14 | 2019-06-14 | A kind of whole blood DNA extracts kit can be used for building library |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112553193A (en) * | 2020-12-23 | 2021-03-26 | 苏州中科先进技术研究院有限公司 | Kit for extracting whole blood DNA by paramagnetic particle method and use method thereof |
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| CN112553193A (en) * | 2020-12-23 | 2021-03-26 | 苏州中科先进技术研究院有限公司 | Kit for extracting whole blood DNA by paramagnetic particle method and use method thereof |
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Application publication date: 20190827 |