CN118389492A - Kit for extracting genomic DNA of dried blood spots by magnetic bead method and application thereof - Google Patents
Kit for extracting genomic DNA of dried blood spots by magnetic bead method and application thereof Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 81
- 239000011324 bead Substances 0.000 title claims abstract description 74
- 239000008280 blood Substances 0.000 title claims abstract description 59
- 210000004369 blood Anatomy 0.000 title claims abstract description 59
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 98
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 96
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 96
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 68
- 229960000789 guanidine hydrochloride Drugs 0.000 claims abstract description 58
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims abstract description 58
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 54
- 239000006166 lysate Substances 0.000 claims abstract description 47
- 238000000605 extraction Methods 0.000 claims abstract description 27
- SYELZBGXAIXKHU-UHFFFAOYSA-N dodecyldimethylamine N-oxide Chemical compound CCCCCCCCCCCC[N+](C)(C)[O-] SYELZBGXAIXKHU-UHFFFAOYSA-N 0.000 claims abstract description 24
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 72
- 238000005406 washing Methods 0.000 claims description 71
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 68
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 54
- 239000007788 liquid Substances 0.000 claims description 36
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 35
- 239000003480 eluent Substances 0.000 claims description 34
- 239000011780 sodium chloride Substances 0.000 claims description 34
- 239000013504 Triton X-100 Substances 0.000 claims description 20
- 229920004890 Triton X-100 Polymers 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 229940124447 delivery agent Drugs 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000004907 flux Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 51
- 230000000052 comparative effect Effects 0.000 description 21
- 239000000203 mixture Substances 0.000 description 13
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000003344 environmental pollutant Substances 0.000 description 3
- 150000002357 guanidines Chemical class 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
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- 238000012216 screening Methods 0.000 description 3
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- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
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- 206010010099 Combined immunodeficiency Diseases 0.000 description 1
- 206010011878 Deafness Diseases 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 201000011252 Phenylketonuria Diseases 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 208000002903 Thalassemia Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
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- 208000016354 hearing loss disease Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 238000007886 magnetic bead extraction Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
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- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
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- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
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- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- 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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Abstract
The invention discloses a kit for extracting genomic DNA of dried blood spots by a magnetic bead method and application thereof. According to the invention, naOH and guanidine hydrochloride are added into the nucleic acid releasing agent, so that cells can be rapidly lysed to release nucleic acid, and meanwhile, the purity of the nucleic acid is obviously improved by adding DTT into the nucleic acid releasing agent. In addition, the concentration and purity of the nucleic acid can be improved by adding lauryl dimethyl amine oxide into the lysate. The kit can efficiently extract nucleic acid in the dried blood spot sample, has the advantages of high purity, high yield, simple operation, short time, full automation, high flux, low cost and the like, and can meet the extraction requirements of a large number of samples.
Description
Technical Field
The invention relates to the technical field of nucleic acid extraction, in particular to a kit for extracting genomic DNA of dried blood spots by a magnetic bead method and application thereof.
Background
The Dry Blood Spot (DBS) is a blood collection mode for collecting a whole blood sample on filter paper, has the advantages of less blood volume requirement, convenient blood sample collection, storage and transportation and the like compared with the traditional blood collection method, is an effective tool in the process of gene detection, especially large-scale genetic disease screening, and has been used for neonatal screening of phenylketonuria in the fifth sixty of the last century. With the development of tandem mass spectrometry technology and the like, dry blood spots are widely applied to 'new screens' of various neonatal genetic metabolic diseases. In recent years, dry blood spots have also been developed in many aspects in the field of gene detection, such as deafness, thalassemia, and the like. In addition, screening of T cell receptor rearrangement deletion loops (TRECs) and Kappa deletion recombination excision loops (KRECs) in neonatal Severe Combined Immunodeficiency (SCID) also used dry blood spots.
The current methods for extracting genomic DNA of the dried blood spots mainly comprise 3 methods: (1) nucleic acid releasing method: washing or not washing the dried blood slices, adding a nucleic acid releasing agent for soaking, performing high-temperature pyrolysis, and centrifuging to obtain nucleic acid; (2) centrifugal column method: the nucleic acid lysate lyses cells in the dried blood slices, and the nucleic acid is released and then is specifically adsorbed by using a silica gel membrane, and then the nucleic acid is eluted to obtain purer nucleic acid; (3) magnetic bead method: the nucleic acid lysate lyses cells in the dried blood, and after releasing nucleic acid, the magnetic beads are used for adsorbing the nucleic acid, and when the conditions are changed, the magnetic beads release the adsorbed nucleic acid, so that the relatively pure nucleic acid is obtained by elution. In recent years, the magnetic bead method has been attracting attention because of its capability of realizing high-throughput automated operation, short time, simple operation, safety, no toxicity, low cost, and the like.
The Chinese patent No. 105602942A discloses a nucleic acid releasing agent and a rapid extraction method of dried blood spot nucleic acid, the nucleic acid releasing agent provided by the method can realize the release of nucleic acid, and the nucleic acid with higher purity is obtained through a filter column, so that the operations of repeated centrifugation, supernatant discarding, tube rotating and the like in the traditional extraction technology are avoided, but the releasing agent needs to be stored in an environment of-20 ℃, which is not beneficial to long-term storage and transportation, and the operation time of the whole experimental process is as long as 4h, which is not beneficial to rapid high-flux nucleic acid extraction.
The invention patent CN113122534A discloses a kit for extracting genomic DNA of dried blood spots by a magnetic bead method and an extraction method, and the reagent for extracting the dried blood spots can extract genomic DNA with complete fragments, stable quality and clean strips, but cannot realize automatic high-flux extraction, and has great influence on the quality of extracting the nucleic acid of the dried blood spots by manual operation.
Chinese patent No. 117165575A discloses a kit for automatically extracting dry blood spot DNA based on nano magnetic beads, which does not contain toxic components such as phenol chloroform and the like, does not need time-consuming alcohol precipitation, and can perform high-flux extraction. However, the average value of A260/A230 of the nucleic acid extracted by the kit is about 1.16 and is far smaller than 2.0, which indicates that the dry blood spot genome DNA extracted by the kit has pollutants such as carbohydrate, guanidine salt and the like, so that the DNA purity of the kit still needs to be further improved.
In summary, the prior art still has some drawbacks in terms of extraction of genomic DNA of dried blood spots, so it is necessary to develop a method that can be suitable for machine-automated extraction while improving concentration and purity, thereby realizing large-scale rapid extraction of samples.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the invention provides a kit for extracting genomic DNA of dried blood spots by a magnetic bead method and application thereof.
A first object of the present invention is to provide a nucleic acid releasing agent for extracting genomic DNA of dried blood spots by a magnetic bead method.
A second object of the present invention is to provide a lysate for extracting genomic DNA of dried blood spots by a magnetic bead method.
A third object of the present invention is to provide the use of the above-mentioned nucleic acid releasing agent and lysate in the preparation of a kit for extracting genomic DNA of dried blood spots by a magnetic bead method.
The fourth object of the invention is to provide a kit for extracting genomic DNA of dried blood spots by a magnetic bead method.
Accordingly, the present invention claims the following:
A nucleic acid delivery agent for use in the magnetic bead extraction of genomic DNA from dried blood spots, said nucleic acid delivery agent comprising the following components: 4 to 10M guanidine hydrochloride, 10 to 50 mM Tris-HCl, 50 to 150 mM NaOH, 10 to 40mM EDTA, triton X-100 with a volume fraction of 0.8 to 1.2 percent and 0.8 to 1.2M DTT.
Preferably, the nucleic acid releasing agent contains the following components: 6 to 8M guanidine hydrochloride, 40 to 50mM Tris-HCl, 110 to 125 mM NaOH, 10 to 15 mM EDTA, 0.8 to 1.2 percent of Triton X-100 and 0.8 to 1.2M DTT.
More preferably, the nucleic acid releasing agent contains the following components: 7M guanidine hydrochloride, 50 mM Tris-HCl, 120 mM NaOH, 10 mM EDTA, 1% by volume Triton X-100 and 1M DTT.
A lysate for extracting genomic DNA of dried blood spots by a magnetic bead method, the lysate comprising the following components: 1 to 5M guanidine hydrochloride, 40 to 50mM Tris-HCl, 10 to 40 mM EDTA, 1 to 3M sodium chloride and 1 to 2 percent of lauryl dimethyl amine oxide by volume fraction.
Preferably, the lysate contains the following components: 3M guanidine hydrochloride, 50 mM Tris-HCl, 20 mM EDTA, 3M sodium chloride and 1.5% by volume of lauryl dimethyl amine oxide.
The application of any one of the nucleic acid releasing agent and/or the lysate in preparing a product for extracting genomic DNA of dried blood spots by a magnetic bead method.
A kit for extracting genomic DNA of dried blood spots by a magnetic bead method, wherein the kit contains any one of the nucleic acid releasing agents and/or the lysis solution.
Preferably, the kit further comprises magnetic beads.
More preferably, the magnetic beads are silica hydroxyl magnetic beads.
More preferably, the concentration of the magnetic beads is 1.8-2.2 mg/mL.
Most preferably, the concentration of the magnetic beads is 2 mg/mL.
Preferably, the kit also contains a washing liquid I;
the washing liquid I contains the following components: 1 to 3M guanidine hydrochloride, 0.5 to 0.8M sodium chloride, 25 to 35 mM Tris-HCl and 20 to 25 mM EDTA.
More preferably, the washing liquid i contains the following components: 1M guanidine hydrochloride, 0.8M sodium chloride, 30 mM Tris-HCl and 25 mM EDTA.
Preferably, the kit also contains a washing liquid II, wherein the washing liquid II is ethanol solution with the volume fraction of 70-75%.
More preferably, the washing liquid II is ethanol solution with the volume fraction of 70%.
Preferably, the kit also contains an eluent, wherein the eluent is 8-10 mM Tris-HCl or RNase-free water.
More preferably, the eluent is 10 mM Tris-HCl or RNase-free water.
More preferably, the pH of the 10 mM Tris-HCl is 8.0.
Compared with the prior art, the invention has the following beneficial effects:
The invention discloses a kit for extracting genomic DNA of dried blood spots by a magnetic bead method and application thereof. The kit consists of a nucleic acid releasing agent, a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent, wherein the nucleic acid releasing agent comprises the following components: guanidine hydrochloride, tris-HCl, naOH, EDTA, triton X-100 and DTT; the lysate comprises the following components: guanidine hydrochloride, tris-HCl, EDTA, sodium chloride and lauryl dimethyl amine oxide; the magnetic beads are silicon hydroxyl magnetic beads; the washing liquid I comprises the following components: guanidine hydrochloride, sodium chloride, tris-HCl and EDTA; the washing solution II is ethanol solution, and the eluent is Tris-HCl or RNase-free water.
Compared with the existing products, the kit provided by the invention has at least the following advantages:
(1) The nucleic acid releasing agent can quickly lyse cells by the combined action of a strong protein lysing agent and a strong alkaline substance to release nucleic acid, and the purity of the nucleic acid is obviously improved by adding DTT in a strong alkaline solution state;
(2) The extraction procedure of the invention adopts the method of adsorbing the nucleic acid to the magnetic beads while cracking, and can greatly reduce the extraction time under the condition of ensuring the purity and the concentration of the extracted nucleic acid.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Silica-hydroxyl magnetic beads: purchased from Shenzhen Maifu New Material technologies Co., ltd., product number: SM200H, particle size: 200 nm.
Example 1A kit for extracting genomic DNA of dried blood spots by magnetic bead method
1. Composition of components
The kit comprises a nucleic acid releasing agent, a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The nucleic acid releasing agent comprises the following components: 4M guanidine hydrochloride, 10 mM Tris-HCl, 50 mM NaOH, 10 mM EDTA, and 1% Triton X-100 by volume fraction.
The lysate comprises the following components: 3M guanidine hydrochloride, 40 mM Tris-HCl, 10 mM EDTA, 1M sodium chloride, 1.5% by volume lauryl dimethyl amine oxide.
The concentration of the silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 1M guanidine hydrochloride, 0.5M sodium chloride, 30 mM Tris-HCl, 20 mM EDTA.
The washing liquid II comprises the following components: ethanol solution with volume fraction of 70%.
The eluent was 10 mM Tris-HCl at pH 8.0, or RNase-free water.
2. Application method
1. And (3) sub-packaging each component in the kit into a pre-packaging reagent plate of a 96-well plate, wherein the 1 st column and the 7 th column of the pre-packaging reagent plate are internally provided with a lysate, the 2 nd column and the 8 th column of the deep-hole plate are internally provided with a washing liquid I, the 3 rd column and the 9 th column of the deep-hole plate are internally provided with a washing liquid II, the 5 th column and the 11 th column of the deep-hole plate are internally provided with a magnetic bead solution, and the 6 th column and the 12 th column of the deep-hole plate are internally provided with an eluent. Specifically, the results are shown in Table 1.
Table 1 pre-packed reagent plate
2. Taking 1 round hole dry blood spot sample with diameter of 6 mm into a centrifuge tube (self-contained) of 1.5 mL, adding 300 mu L of nucleic acid releasing agent, adding 20 mu L of proteinase K solution, mixing uniformly by vortex oscillation 10 s, putting into a constant temperature oscillator preheated to 56 ℃, oscillating 10 min at constant temperature of 900 rpm, and then centrifuging briefly to obtain supernatant as an extraction sample.
2. The extracted samples were added at columns 1 and 7, respectively.
3. Opening a Kaipu full-automatic nucleic acid extractor HBNP-4801A and the same type of instruments, putting a pre-packed reagent plate added with a sample into the extractor, and then inserting a magnetic rod sleeve.
4. The corresponding nucleic acid extraction program was selected and the extraction program was run, as shown in tables 2 and 3, with both extraction programs running simultaneously.
Table 2 extraction procedure 1
TABLE 3 extraction procedure 2
5. After the automatic procedure is finished, the pre-packaged reagent plate and the magnetic sleeve are taken out, and the nucleic acid solution obtained by extraction in the 6 th column and the 12 th column is taken out for further detection or stored at-20+/-5 ℃.
6. A2 mu L nucleic acid solution is adopted to carry out fluorescence spectrophotometry detection by using Nanodrop 2000 to obtain the ratio of A260/A280 and A260/A230, the ratio of A260/A280 is less than or equal to 1.8 and less than or equal to 2.0 of a pure DNA sample, if the ratio is less than 1.8, the influence of protein or phenolic substances exists, the ratio of A260/A230 is more than 2.0, and if the ratio is less than 2.0, the existence of pollutants such as carbohydrate, guanidine salt and the like in the sample is indicated.
Example 2A kit for extracting genomic DNA of dried blood spots by magnetic bead method
1. Composition of components
The kit comprises a nucleic acid releasing agent, a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The nucleic acid releasing agent comprises the following components: 4M guanidine hydrochloride, 40 mM Tris-HCl, 70 mM NaOH, 10 mM EDTA, triton X-100 with a volume fraction of 1%.
The lysate comprises the following components: 3M guanidine hydrochloride, 40 mM Tris-HCl, 10mM EDTA, 1M sodium chloride, 1.5% by volume lauryl dimethyl amine oxide.
The concentration of the silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 1M guanidine hydrochloride, 0.5M sodium chloride, 30 mM Tris-HCl, 20 mM EDTA.
The washing liquid II comprises the following components: ethanol solution with volume fraction of 70%.
The eluent was 10 mM Tris-HCl at pH 8.0, or RNase-free water.
2. Application method
The use was made according to the method of use of example 1.
Example 3A kit for extracting genomic DNA of dried blood spots by magnetic bead method
1. Composition of components
The kit comprises a nucleic acid releasing agent, a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The nucleic acid releasing agent comprises the following components: 6M guanidine hydrochloride, 50 mM Tris-HCl, 70 mM NaOH, 25 mM EDTA, and 1% by volume Triton X-100.
The lysate comprises the following components: 1M guanidine hydrochloride, 40 mM Tris-HCl, 10mM EDTA, 1M sodium chloride, 1.5% by volume lauryl dimethyl amine oxide.
The concentration of the silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 1M guanidine hydrochloride, 0.5M sodium chloride, 30 mM Tris-HCl, 20 mM EDTA.
The washing liquid II comprises the following components: ethanol solution with volume fraction of 70%.
The eluent was 10 mM Tris-HCl at pH 8.0, or RNase-free water.
2. Application method
The use was made according to the method of use of example 1.
Example 4A kit for extracting genomic DNA of dried blood spots by magnetic bead method
1. Composition of components
The kit comprises a nucleic acid releasing agent, a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The nucleic acid releasing agent comprises the following components: 7M guanidine hydrochloride, 50mM Tris-HCl, 100mM NaOH, 40mM EDTA, and 1% by volume Triton X-100.
The lysate comprises the following components: 5M guanidine hydrochloride, 50mM Tris-HCl, 40 mM EDTA, 3M sodium chloride, 1.5% by volume lauryl dimethyl amine oxide.
The concentration of the silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 2M guanidine hydrochloride, 0.8M sodium chloride, 30 mM Tris-HCl, 20 mM EDTA.
The washing liquid II comprises the following components: ethanol solution with volume fraction of 70%.
The eluent was 10 mM Tris-HCl at pH 8.0, or RNase-free water.
2. Application method
The use was made according to the method of use of example 1.
Example 5A kit for extracting genomic DNA of dried blood spots by magnetic bead method
1. Composition of components
The kit comprises a nucleic acid releasing agent, a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The nucleic acid releasing agent comprises the following components: 7M guanidine hydrochloride, 50mM Tris-HCl, 120 mM NaOH, 10mM EDTA, 1% by volume Triton X-100, 1M DTT.
The lysate comprises the following components: 3M guanidine hydrochloride, 50mM Tris-HCl, 20 mM EDTA, 3M sodium chloride, 1.5% by volume lauryl dimethyl amine oxide.
The concentration of the silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 1M guanidine hydrochloride, 0.8M sodium chloride, 30 mM Tris-HCl, 25mM EDTA.
The washing liquid II comprises the following components: ethanol solution with volume fraction of 70%.
The eluent was 10 mM Tris-HCl at pH 8.0, or RNase-free water.
2. Application method
The procedure used was as in example 1, except that the extraction procedures were run simultaneously as shown in tables 4 and 5.
Table 4 extraction procedure 1
Table 5 extraction procedure 2
Example 6A kit for extracting genomic DNA of dried blood spots by magnetic bead method
1. Composition of components
The kit comprises a nucleic acid releasing agent, a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The nucleic acid releasing agent comprises the following components: 7M guanidine hydrochloride, 50mM Tris-HCl, 120 mM NaOH, 40mM EDTA, and 1% by volume Triton X-100.
The lysate comprises the following components: 5M guanidine hydrochloride, 50mM Tris-HCl, 20mM EDTA, 3M sodium chloride, 1.5% by volume lauryl dimethyl amine oxide.
The concentration of the silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 1M guanidine hydrochloride, 0.8M sodium chloride, 30 mM Tris-HCl, 25mM EDTA.
The washing liquid II comprises the following components: ethanol solution with volume fraction of 70%.
The eluent was 10 mM Tris-HCl at pH 8.0, or RNase-free water.
2. Application method
The use was made according to the method of use of example 1.
Example 7A kit for extracting genomic DNA of dried blood spots by magnetic bead method
1. Composition of components
The kit comprises a nucleic acid releasing agent, a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The nucleic acid releasing agent comprises the following components: 10M guanidine hydrochloride, 50 mM Tris-HCl, 150 mM NaOH, 40mM EDTA, triton X-100 with a volume fraction of 1%.
The lysate comprises the following components: 4M guanidine hydrochloride, 50mM Tris-HCl, 20mM EDTA, 3M sodium chloride, 1.5% by volume lauryl dimethyl amine oxide.
The concentration of the silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 3M guanidine hydrochloride, 0.8M sodium chloride, 30 mM Tris-HCl, 25mM EDTA.
The washing liquid II comprises the following components: ethanol solution with volume fraction of 70%.
The eluent was 10 mM Tris-HCl at pH 8.0, or RNase-free water.
2. Application method
The use was made according to the method of use of example 1.
Example 8A kit for extracting genomic DNA of dried blood spots by magnetic bead method
1. Composition of components
The kit comprises a nucleic acid releasing agent, a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The nucleic acid releasing agent comprises the following components: 4M guanidine hydrochloride, 50 mM Tris-HCl, 120mM NaOH, 10mM EDTA, 1% by volume Triton X-100.
The lysate comprises the following components: 3M guanidine hydrochloride, 50mM Tris-HCl, 20 mM EDTA, 3M sodium chloride, 1.5% by volume lauryl dimethyl amine oxide.
The concentration of the silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 1M guanidine hydrochloride, 0.8M sodium chloride, 30 mM Tris-HCl, 25mM EDTA.
The washing liquid II comprises the following components: ethanol solution with volume fraction of 70%.
The eluent was 10 mM Tris-HCl at pH 8.0, or RNase-free water.
2. Application method
The use was made according to the method of use of example 1.
Example 9A kit for extracting genomic DNA of dried blood spots by magnetic bead method
1. Composition of components
The kit comprises a nucleic acid releasing agent, a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The nucleic acid releasing agent comprises the following components: 5M guanidine hydrochloride, 50 mM Tris-HCl, 120mM NaOH, 10mM EDTA, and 1% by volume Triton X-100.
The lysate comprises the following components: 3M guanidine hydrochloride, 50mM Tris-HCl, 20 mM EDTA, 3M sodium chloride, 1.5% by volume lauryl dimethyl amine oxide.
The concentration of the silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 1M guanidine hydrochloride, 0.8M sodium chloride, 30 mM Tris-HCl, 25mM EDTA.
The washing liquid II comprises the following components: ethanol solution with volume fraction of 70%.
The eluent was 10 mM Tris-HCl at pH 8.0, or RNase-free water.
2. Application method
The use was made according to the method of use of example 1.
Example 10A kit for extracting genomic DNA of dried blood spots by magnetic bead method
1. Composition of components
The kit comprises a nucleic acid releasing agent, a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The nucleic acid releasing agent comprises the following components: 9M guanidine hydrochloride, 50 mM Tris-HCl, 120mM NaOH, 10mM EDTA, and 1% by volume Triton X-100.
The lysate comprises the following components: 3M guanidine hydrochloride, 50mM Tris-HCl, 20 mM EDTA, 3M sodium chloride, 1.5% by volume lauryl dimethyl amine oxide.
The concentration of the silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 1M guanidine hydrochloride, 0.8M sodium chloride, 30 mM Tris-HCl, 25mM EDTA.
The washing liquid II comprises the following components: ethanol solution with volume fraction of 70%.
The eluent was 10 mM Tris-HCl at pH 8.0, or RNase-free water.
2. Application method
The use was made according to the method of use of example 1.
Example 11A kit for extracting genomic DNA from dried blood spots by magnetic bead method
1. Composition of components
The kit comprises a nucleic acid releasing agent, a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The nucleic acid releasing agent comprises the following components: 7M guanidine hydrochloride, 50 mM Tris-HCl, 50 mM NaOH, 10 mM EDTA, and 1% by volume Triton X-100.
The lysate comprises the following components: 3M guanidine hydrochloride, 50mM Tris-HCl, 20 mM EDTA, 3M sodium chloride, 1.5% by volume lauryl dimethyl amine oxide.
The concentration of the silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 1M guanidine hydrochloride, 0.8M sodium chloride, 30 mM Tris-HCl, 25mM EDTA.
The washing liquid II comprises the following components: ethanol solution with volume fraction of 70%.
The eluent was 10 mM Tris-HCl at pH 8.0, or RNase-free water.
2. Application method
The procedure of example 5 was followed.
Example 12A kit for extracting genomic DNA of dried blood spots by magnetic bead method
1. Composition of components
The kit comprises a nucleic acid releasing agent, a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The nucleic acid releasing agent comprises the following components: 7M guanidine hydrochloride, 50 mM Tris-HCl, 90 mM NaOH, 10 mM EDTA, and 1% by volume Triton X-100.
The lysate comprises the following components: 3M guanidine hydrochloride, 50mM Tris-HCl, 20 mM EDTA, 3M sodium chloride, 1.5% by volume lauryl dimethyl amine oxide.
The concentration of the silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 1M guanidine hydrochloride, 0.8M sodium chloride, 30 mM Tris-HCl, 25mM EDTA.
The washing liquid II comprises the following components: ethanol solution with volume fraction of 70%.
The eluent was 10 mM Tris-HCl at pH 8.0, or RNase-free water.
2. Application method
The procedure of example 5 was followed.
Example 13A kit for extracting genomic DNA of dried blood spots by magnetic bead method
1. Composition of components
The kit comprises a nucleic acid releasing agent, a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The nucleic acid releasing agent comprises the following components: 7M guanidine hydrochloride, 50mM Tris-HCl, 150 mM NaOH, 10mM EDTA, and 1% by volume Triton X-100.
The lysate comprises the following components: 3M guanidine hydrochloride, 50mM Tris-HCl, 20 mM EDTA, 3M sodium chloride, 1.5% by volume lauryl dimethyl amine oxide.
The concentration of the silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 1M guanidine hydrochloride, 0.8M sodium chloride, 30 mM Tris-HCl, 25mM EDTA.
The washing liquid II comprises the following components: ethanol solution with volume fraction of 70%.
The eluent was 10 mM Tris-HCl at pH 8.0, or RNase-free water.
2. Application method
The procedure of example 5 was followed.
Comparative example 1
The difference between this comparative example and example 5 is that guanidine hydrochloride is not contained in the nucleic acid releasing agent, and the other is the same as in example 5.
Comparative example 2
The difference between this comparative example and example 5 is that the nucleic acid releasing agent does not contain NaOH, and the other is the same as in example 5.
Comparative example 3
The difference between this comparative example and example 5 is that the nucleic acid releasing agent does not contain NaOH or guanidine hydrochloride, and the other is the same as in example 5.
Comparative example 4
The difference between this comparative example and example 5 is that the nucleic acid releasing agent does not contain DTT, and the other is the same as in example 5.
Comparative example 5
The difference between this comparative example and example 5 is that the lysate does not contain lauryl dimethyl amine oxide, otherwise the same as in example 5.
Comparative example 6
The difference between this comparative example and example 5 is that lauryl dimethyl amine oxide in the lysate was replaced with tween 20 in the same volume percentage, otherwise as in example 5.
Comparative example 7
The difference between this comparative example and example 5 is that lauryl dimethyl amine oxide in the lysate was replaced with tween 80 in the same volume percentage, otherwise as in example 5.
Comparative example 8
This comparative example and example 5 were different in that the extraction procedure shown in tables 2 and 3 was followed, and the procedure was otherwise identical to that of example 5.
Performance testing
Nucleic acids were extracted from the same blood dry blood spot samples using the kits of examples 1 to 13 and comparative examples 1 to 8, and the concentration and purity of the extracted nucleic acids are shown in Table 6.
TABLE 6 nucleic acid concentration and purity
As can be seen from the data in Table 6, the kit of examples 1 to 13 of the present invention was used to sufficiently lyse the lysate and sufficiently clean the washing solution, thereby finally achieving a high yield and a good purity of nucleic acids. In particular, the kit and the method provided in example 5 have the highest concentration and the best purity of the dry blood spot genome DNA, which show that the components in example 5 fully play a synergistic effect, and the extraction method is matched with the kit and the method to jointly realize high DNA concentration, small influence of protein or phenolic substances and few pollutants such as guanidine salt.
As can be seen from the data of example 5 and comparative examples 1 to 3, the addition of guanidine hydrochloride and NaOH to the nucleic acid releasing agent can fully exert the synergistic effect, enhance cleavage and increase not only the concentration of extracted nucleic acid but also the purity of nucleic acid.
As can be seen from the data of example 5 and comparative example 4, the ratio of A260/A230 of the extracted dried blood spot nucleic acid is obviously improved after the synergist DTT is added.
As is clear from the data of example 5 and comparative examples 5 to 7, the concentration and purity of the extracted dried blood spot nucleic acid were low without adding lauryldimethyl amine oxide or with substituting lauryldimethyl amine oxide for Tween 20 and Tween 80 in the lysate, and only adding lauryldimethyl amine oxide had the best effect on the concentration and purity of the extracted nucleic acid.
As can be seen from the data results of example 5 and comparative example 8, the reduction of the binding moiety in the extraction procedure not only shortens the extraction period, but also does not reduce the concentration and purity of the extracted dried blood spot nucleic acid while improving the extraction efficiency.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A nucleic acid delivery agent for extracting genomic DNA from dried blood spots by a magnetic bead method, the nucleic acid delivery agent comprising the following components: 4 to 10M guanidine hydrochloride, 10 to 50 mM Tris-HCl, 50 to 150 mM NaOH, 10 to 40 mM EDTA, triton X-100 with a volume fraction of 0.8 to 1.2 percent and 0.8 to 1.2M DTT.
2. The nucleic acid releasing agent of claim 1, wherein the nucleic acid releasing agent contains the following components: 6 to 8M guanidine hydrochloride, 40 to 50 mM Tris-HCl, 110 to 125 mM NaOH, 10 to 15 mM EDTA, 0.8 to 1.2 percent of Triton X-100 and 0.8 to 1.2M DTT.
3. The nucleic acid releasing agent of claim 2, wherein the nucleic acid releasing agent contains the following components: 7M guanidine hydrochloride, 50 mM Tris-HCl, 120 mM NaOH, 10 mM EDTA, 1% by volume Triton X-100 and 1M DTT.
4. A lysate for extracting genomic DNA of dried blood spots by a magnetic bead method, the lysate comprising the following components: 1 to 5M guanidine hydrochloride, 40 to 50 mM Tris-HCl, 10 to 40 mM EDTA, 1 to 3M sodium chloride and 1 to 2 percent of lauryl dimethyl amine oxide by volume fraction.
5. Use of a nucleic acid releasing agent according to any one of claims 1 to 3 and/or a lysate according to claim 4 for the preparation of a product for the extraction of genomic DNA of dried blood spots by the magnetic bead method.
6. A kit for extracting genomic DNA of a dried blood spot by a magnetic bead method, wherein the kit comprises the nucleic acid releasing agent according to any one of claims 1 to 3 and/or the lysate according to claim 4.
7. The kit of claim 6, further comprising magnetic beads.
8. The kit according to claim 6, wherein the kit further comprises a washing solution I;
the washing liquid I contains the following components: 1 to 3M guanidine hydrochloride, 0.5 to 0.8M sodium chloride, 25 to 35 mM Tris-HCl and 20 to 25 mM EDTA.
9. The kit according to claim 6, further comprising a washing solution II, wherein the washing solution II is an ethanol solution with a volume fraction of 70-75%.
10. The kit according to claim 6, further comprising an eluent, wherein the eluent is 8-10 mM Tris-HCl or RNase-free water.
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