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CN117165575A - Kit for automatically extracting dry blood spot DNA based on nano magnetic beads - Google Patents

Kit for automatically extracting dry blood spot DNA based on nano magnetic beads Download PDF

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Publication number
CN117165575A
CN117165575A CN202311093777.2A CN202311093777A CN117165575A CN 117165575 A CN117165575 A CN 117165575A CN 202311093777 A CN202311093777 A CN 202311093777A CN 117165575 A CN117165575 A CN 117165575A
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CN
China
Prior art keywords
concentration
kit
magnetic beads
aminomethane
automatically extracting
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202311093777.2A
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Chinese (zh)
Inventor
陈荷萍
蚁珩鑫
李娇
黄嘉琪
林程忠
黄亮
陈凯婷
刘晓倩
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Guangzhou Bay Area Biotechnology Co ltd
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Guangzhou Bay Area Biotechnology Co ltd
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Application filed by Guangzhou Bay Area Biotechnology Co ltd filed Critical Guangzhou Bay Area Biotechnology Co ltd
Priority to CN202311093777.2A priority Critical patent/CN117165575A/en
Publication of CN117165575A publication Critical patent/CN117165575A/en
Pending legal-status Critical Current

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    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to a kit for automatically extracting DNA of a dried blood spot based on nano magnetic beads, belonging to the technical field of molecular biology. Comprises a lysis solution AL, proteinase K, a lysis binding solution BLN, magnetic beads MBVB, a washing solution DW1, a washing solution DW2 and an eluting solution DE. The invention provides a kit for automatically extracting DNA of dried blood spots based on nano magnetic beads, which is based on a nano magnetic bead adsorption nucleic acid technology, purifies high-quality nucleic acid from a dried blood spot sample, does not contain toxic components such as phenol and chloroform, does not need time-consuming alcohol precipitation, can perform high-flux extraction experiments by integrating a magnetic rod method or a pipetting automatic nucleic acid extractor, can protect the safety of experimental staff while improving the extraction efficiency, and can be manually operated by matching a magnetic separation frame, and is compatible with the automatic method and the manual method for extraction.

Description

Kit for automatically extracting dry blood spot DNA based on nano magnetic beads
Technical Field
The invention relates to a kit for automatically extracting DNA of a dried blood spot based on nano magnetic beads, belonging to the technical field of molecular biology.
Background
The extraction of genomic nucleic acid from dried blood spots is a very common requirement, and the extraction of the genome of lymphocytes in dried blood spots can be applied to clinical medical disease diagnosis and adjuvant therapy through the analysis of the genome. At present, the dry blood spot genome DNA extraction mainly comprises a column membrane method and a magnetic bead method, and compared with a saturated phenol-chloroform method, a salting-out precipitation method and the like, the magnetic bead method does not need toxic phenol-chloroform extraction or time-consuming alcohol precipitation in the extraction process, so that the safety of operators is ensured; furthermore, the column membrane extraction cannot be used in machines nor in high throughput operation by automated methods, and the extraction efficiency is general.
Disclosure of Invention
In view of the above, the invention provides a kit for automatically extracting dry blood spot DNA based on nano magnetic beads, which is based on a nano magnetic bead adsorption nucleic acid technology, purifies high-quality nucleic acid from a dry blood spot sample, does not contain toxic components such as phenol and chloroform, does not need time-consuming alcohol precipitation, can perform high-flux extraction experiments by integrating a magnetic rod method or a pipetting method automatic nucleic acid extractor, can protect the safety of experimental staff while improving the extraction efficiency, and can be manually operated by matching a magnetic separation frame, and is compatible with the automatic method and the manual method.
The invention provides a kit for automatically extracting dry blood spot DNA (deoxyribonucleic acid) based on nano magnetic beads, which comprises a lysate AL, proteinase K, a lysate BLN, magnetic beads MBVB, a washing solution DW1, a washing solution DW2 and an eluent DE;
the lysate AL comprises tris (hydroxymethyl) aminomethane, ethylenediamine tetraacetic acid, sodium chloride and sodium dodecyl sulfate;
the lysis binding solution BLN comprises tris (hydroxymethyl) aminomethane, guanidine thiocyanate, sodium citrate and triton X-100;
the magnetic beads MBVB are polystyrene coated silicon hydroxyl magnetic beads;
the washing liquid DW1 comprises guanidine thiocyanate, ethylenediamine tetraacetic acid, sodium citrate, sodium chloride and absolute ethyl alcohol;
the washing liquid DW2 comprises tris (hydroxymethyl) aminomethane, sodium chloride and absolute ethyl alcohol;
the eluent DE comprises tris (hydroxymethyl) aminomethane and ethylenediamine tetraacetic acid.
Preferably, the concentration of the tris (hydroxymethyl) aminomethane in the pyrolysis liquid AL is 20-80mmol/L, the concentration of the ethylenediamine tetraacetic acid is 5-30mmol/L, the concentration of the sodium chloride is 50-300mmol/L, and the concentration of the sodium dodecyl sulfate is 0.1% -2%.
Preferably, the concentration of the tris (hydroxymethyl) aminomethane in the lysis binding solution BLN is 20-80mmol/L, the concentration of guanidine thiocyanate is 2.5-5mol/L, the concentration of sodium citrate is 0.1-2%, the concentration of triton X-100 is 5-15%, and HCL and NaOH are used for adjusting the PH to be 5-10.
Preferably, the concentration of ethylenediamine tetraacetic acid in the washing solution DW1 is 1-10mmol/L, the concentration of sodium citrate is 0.1-2%, the concentration of sodium chloride is 10-200mmol/L, and the concentration of absolute ethyl alcohol is 30-80%.
Preferably, the concentration of the tris (hydroxymethyl) aminomethane in the washing solution DW2 is 20-80mmol/L, the concentration of the sodium chloride is 10-200mmol/L, and the concentration of the absolute ethyl alcohol is 30% -80%.
Preferably, the concentration of the tris (hydroxymethyl) aminomethane in the eluent DE is 10-50mmol/L, the concentration of the ethylenediamine tetraacetic acid is 1-10mmol/L, and HCL and NaOH are used for adjusting the PH to be 5-10.
Preferably, the kit also comprises blood sheets and centrifuge tubes.
The invention has the beneficial effects that:
the invention provides a kit for automatically extracting dry blood spot DNA (deoxyribonucleic acid) based on a nano magnetic bead adsorption nucleic acid technology, which is used for purifying high-quality nucleic acid from a dry blood spot sample, does not contain toxic components such as phenol and chloroform, does not need time-consuming alcohol precipitation, can perform high-flux extraction experiments by integrating a magnetic rod method or a pipetting automatic nucleic acid extractor, can realize automatic and high-flux operation, can simultaneously realize treatment of a plurality of samples in the previous extraction time of one sample by matching with the 96/192/384-hole automatic nucleic acid extractor, meets the requirement of the current biological high-flux operation, can protect the safety of experimental staff while improving the extraction efficiency, and can perform manual operation by matching with a magnetic separation frame, and is compatible with the automatic and manual extraction.
Drawings
FIG. 1 is an agarose gel diagram of a kit for automatically extracting DNA of dried blood spots based on nano magnetic beads.
Detailed Description
Preferred embodiments of the present invention will be described in detail below.
The invention provides a kit for automatically extracting dry blood spot DNA (deoxyribonucleic acid) based on nano magnetic beads, which comprises a lysate AL, proteinase K, a lysate BLN, magnetic beads MBVB, a washing solution DW1, a washing solution DW2 and an eluent DE;
the pyrolysis liquid AL comprises tris (hydroxymethyl) aminomethane, ethylenediamine tetraacetic acid, sodium chloride and sodium dodecyl sulfate, wherein the concentration of the tris (hydroxymethyl) aminomethane in the pyrolysis liquid AL is 20-80mmol/L, the concentration of the ethylenediamine tetraacetic acid is 5-30mmol/L, the concentration of the sodium chloride is 50-300mmol/L, and the concentration of the sodium dodecyl sulfate is 0.1% -2%;
the cracking combination solution BLN comprises tris (hydroxymethyl) aminomethane, guanidine thiocyanate, sodium citrate and triton X-100, wherein the concentration of the tris (hydroxymethyl) aminomethane in the cracking combination solution BLN is 20-80mmol/L, the concentration of the guanidine thiocyanate is 2.5-5mol/L, the concentration of the sodium citrate is 0.1-2%, the concentration of the triton X-100 is 5-15%, and HCL and NaOH are used for adjusting the PH to be 5-10;
the magnetic beads MBVB are polystyrene coated silicon hydroxyl magnetic beads;
the washing liquid DW1 comprises guanidine thiocyanate, ethylene diamine tetraacetic acid, sodium citrate, sodium chloride and absolute ethyl alcohol, wherein the concentration of the ethylene diamine tetraacetic acid in the washing liquid DW1 is 1-10mmol/L, the concentration of the sodium citrate is 0.1-2%, the concentration of the sodium chloride is 10-200mmol/L, and the concentration of the absolute ethyl alcohol is 30-80%;
the washing solution DW2 comprises tris (hydroxymethyl) aminomethane, sodium chloride and absolute ethyl alcohol, wherein the concentration of the tris (hydroxymethyl) aminomethane in the washing solution DW2 is 20-80mmol/L, the concentration of the sodium chloride is 10-200mmol/L, and the concentration of the absolute ethyl alcohol is 30-80%;
the eluent DE comprises tris (hydroxymethyl) aminomethane and ethylenediamine tetraacetic acid, wherein the concentration of the tris (hydroxymethyl) aminomethane in the eluent DE is 10-50mmol/L, the concentration of the ethylenediamine tetraacetic acid is 1-10mmol/L, and HCL and NaOH are used for adjusting the PH to 5-10.
The invention also comprises a blood tablet and a centrifuge tube.
The application method of the invention comprises the following steps:
1) Punching 3-5 blood slices with the diameter of 3mm on the FTA Card or the blood slices by using a puncher or cutting the blood slices, and transferring the blood slices into a 1.5mL or 2.0mL centrifuge tube;
2) Adding 400 mu L of lysate AL and 20 mu L of proteinase K into the centrifuge tube, mixing for 30 seconds by high-speed vortex on a vortex instrument, and checking to ensure that blood chips are immersed in the solution;
3) Oscillating the temperature bath at 60 ℃ for 45 minutes, taking out vortex 1 time every 15 minutes and 30 seconds each time during the temperature bath if the thermostat has no oscillation function;
4) After returning to room temperature, centrifuging to collect the liquid drops of the pipe cover into the pipe;
5) Transferring all the warm bath liquid in the step 4) into a pre-assembled plate cracking binding liquid BLN hole, completing extraction by a nucleic acid extractor operation program, and respectively taking 2ul of the extracted liquid, and measuring the concentration and 260/280,260/230 ratio by a nanodrop spectrophotometer; 2ul of each is taken and subjected to agarose gel electrophoresis to verify DNA extraction;
6) Nucleic acid extractor program settings
Step one The lysate BLN was mixed with the sample for 10 minutes
Step two Magnetic bead
Step three Mixing nucleic acid at room temperature for 5 min, and adsorbing magnetic beads
Step four Washing liquid DW1 is washed for 1 time, and magnetic beads are adsorbed
Step five Washing liquid DW2 washes 1 time, and adsorbs magnetic beads
Step six Washing liquid DW2 washes 1 time, and adsorbs magnetic beads
Step seven Airing for one minute
Step eight Eluting with 70 ℃ eluent DE for 5 minutes to adsorb magnetic beads
For a long time Magnetic bead
7) Results display
Nanodrop spectrophotometer for measuring concentration and 260/280,260/230 ratio
The numbers 1-5 are the genomic DNA of the dried blood spots extracted by the commercial A brand column method.
The number 6-10 is the extraction of the genomic DNA of the dried blood spots by the kit of the invention.
The invention and its embodiments have been described above without limitation, and the actual construction is not limited thereto. In summary, if one of ordinary skill in the art is informed by this disclosure, a structural manner and an embodiment similar to the technical solution should not be creatively devised without departing from the gist of the present invention.

Claims (7)

1. A kit for automatically extracting dry blood spot DNA based on nano magnetic beads is characterized in that: comprises a lysis solution AL, proteinase K, a lysis binding solution BLN, magnetic beads MBVB, a washing solution DW1, a washing solution DW2 and an eluent DE;
the lysate AL comprises tris (hydroxymethyl) aminomethane, ethylenediamine tetraacetic acid, sodium chloride and sodium dodecyl sulfate;
the lysis binding solution BLN comprises tris (hydroxymethyl) aminomethane, guanidine thiocyanate, sodium citrate and triton X-100; the magnetic beads MBVB are polystyrene coated silicon hydroxyl magnetic beads;
the washing liquid DW1 comprises guanidine thiocyanate, ethylenediamine tetraacetic acid, sodium citrate, sodium chloride and absolute ethyl alcohol; the washing liquid DW2 comprises tris (hydroxymethyl) aminomethane, sodium chloride and absolute ethyl alcohol;
the eluent DE comprises tris (hydroxymethyl) aminomethane and ethylenediamine tetraacetic acid.
2. The kit for automatically extracting dry blood spot DNA based on the nano magnetic beads according to claim 1, wherein the kit comprises the following components: the concentration of the tris (hydroxymethyl) aminomethane in the pyrolysis liquid AL is 20-80mmol/L, the concentration of the ethylenediamine tetraacetic acid is 5-30mmol/L, the concentration of the sodium chloride is 50-300mmol/L, and the concentration of the sodium dodecyl sulfate is 0.1% -2%.
3. The kit for automatically extracting dry blood spot DNA based on the nano magnetic beads according to claim 1, wherein the kit comprises the following components: the concentration of the tris (hydroxymethyl) aminomethane in the lysis binding solution BLN is 20-80mmol/L, the concentration of guanidine thiocyanate is 2.5-5mol/L, the concentration of sodium citrate is 0.1-2%, the concentration of triton X-100 is 5-15%, and HCL and NaOH are used for adjusting the PH to be 5-10.
4. The kit for automatically extracting dry blood spot DNA based on the nano magnetic beads according to claim 1, wherein the kit comprises the following components: the concentration of ethylenediamine tetraacetic acid in the washing liquid DW1 is 1-10mmol/L, the concentration of sodium citrate is 0.1% -2%, the concentration of sodium chloride is 10-200mmol/L, and the concentration of absolute ethyl alcohol is 30% -80%.
5. The kit for automatically extracting dry blood spot DNA based on the nano magnetic beads according to claim 1, wherein the kit comprises the following components: the concentration of the tris (hydroxymethyl) aminomethane in the washing liquid DW2 is 20-80mmol/L, the concentration of the sodium chloride is 10-200mmol/L, and the concentration of the absolute ethyl alcohol is 30% -80%.
6. The kit for automatically extracting dry blood spot DNA based on the nano magnetic beads according to claim 1, wherein the kit comprises the following components: the concentration of the trimethylol aminomethane in the eluent DE is 10-50mmol/L, the concentration of the ethylenediamine tetraacetic acid is 1-10mmol/L, and the PH is adjusted to 5-10 by using HCL and NaOH.
7. The kit for automatically extracting dry blood spot DNA based on the nano magnetic beads according to claim 1, wherein the kit comprises the following components: the utility model also comprises a blood tablet and a centrifuge tube.
CN202311093777.2A 2023-08-29 2023-08-29 Kit for automatically extracting dry blood spot DNA based on nano magnetic beads Pending CN117165575A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311093777.2A CN117165575A (en) 2023-08-29 2023-08-29 Kit for automatically extracting dry blood spot DNA based on nano magnetic beads

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311093777.2A CN117165575A (en) 2023-08-29 2023-08-29 Kit for automatically extracting dry blood spot DNA based on nano magnetic beads

Publications (1)

Publication Number Publication Date
CN117165575A true CN117165575A (en) 2023-12-05

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Application Number Title Priority Date Filing Date
CN202311093777.2A Pending CN117165575A (en) 2023-08-29 2023-08-29 Kit for automatically extracting dry blood spot DNA based on nano magnetic beads

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118389492A (en) * 2024-07-01 2024-07-26 广州凯普医药科技有限公司 Kit for extracting genomic DNA of dried blood spots by magnetic bead method and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118389492A (en) * 2024-07-01 2024-07-26 广州凯普医药科技有限公司 Kit for extracting genomic DNA of dried blood spots by magnetic bead method and application thereof
CN118389492B (en) * 2024-07-01 2024-10-15 广州凯普医药科技有限公司 Kit for extracting genomic DNA of dried blood spots by magnetic bead method and application thereof

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