WO2024139530A1 - Kit et procédé d'extraction d'adn dans un échantillon de tissu végétal - Google Patents

Kit et procédé d'extraction d'adn dans un échantillon de tissu végétal Download PDF

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WO2024139530A1
WO2024139530A1 PCT/CN2023/124174 CN2023124174W WO2024139530A1 WO 2024139530 A1 WO2024139530 A1 WO 2024139530A1 CN 2023124174 W CN2023124174 W CN 2023124174W WO 2024139530 A1 WO2024139530 A1 WO 2024139530A1
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dna
buffer
stabilizer
concentration
tris
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PCT/CN2023/124174
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Chinese (zh)
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王洋
李慧
王超
李小康
梁梦蝶
陶庆
赵云龙
梁帆
汪德鹏
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武汉希望组生物科技有限公司
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Publication of WO2024139530A1 publication Critical patent/WO2024139530A1/fr

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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
    • 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

Definitions

  • third-generation single-molecule sequencing has higher requirements on the molecular length and purity of DNA.
  • the ultra-long sequencing requirements of the Oxford Nanopore Sequencing Platform (ONT) are the most stringent. It requires that the extracted and prepared DNA have ultra-high molecular length while maintaining high purity, so as to achieve the maximum sequencing read length and good sequencing data output.
  • Another method for obtaining long DNA fragments is as follows: The plant cells are broken by grinding with liquid nitrogen or homogenizing, and then the broken cells are cleaned with a cleaning solution, and filtered through a filter screen to remove larger impurities to obtain the nucleus. The obtained nucleus is embedded in agarose gel, and then the nucleus embedded in the gel is lysed and digested to release DNA from the nucleus and fix it in the gel to prevent DNA breakage. Then agarose is digested to obtain a DNA solution.
  • kits and methods for extracting DNA from plant tissue samples in the prior art cannot take into account both the length and purity of the DNA extraction product, and thus cannot meet the requirements for ultra-long sequencing of genomic DNA or the ultra-long sequencing requirements of the third-generation sequencing ONT platform.
  • the washing buffer B contains 10-400 mM of a first DNA stabilizer
  • the lysis buffer B contains 10-30% (w/v) anionic surfactant
  • the lysis buffer A and lysis buffer B contained in the extraction kit of the present invention are used in the cell nucleus lysis step, and their main function is to destroy the cell nuclear membrane structure and release DNA through the combined use of the two lysis buffers.
  • the use of the above second metal ion chelators can inhibit the activity of metal-dependent enzymes such as DNA enzymes and RNA enzymes. More preferably, the second metal ion chelator is EDTA. Compared with other metal ion chelators, EDTA can specifically inhibit the activity of DNase and RNase. It will not inhibit the activity of other enzymes.
  • the protease is mainly used in the cell nucleus lysis step.
  • the protease is a reagent that degrades proteins bound to DNA.
  • the protease is proteinase K.
  • Proteinase K is a powerful proteinase isolated from Candida albicans and has high Specific activity is a key reagent in the DNA extraction process. It is used to degrade proteins bound to DNA during DNA extraction, so that the DNA molecules can be separated intact. More preferably, the concentration of proteinase K is 10-25 mg/ml (storage concentration). Proteinase K needs to be diluted when used, and the concentration of the diluted proteinase K is 0.2-0.6 mg/ml (working concentration).
  • RNA enzyme is mainly used in the cell nucleus lysis step, and RNA enzyme is a reagent that plays a role in degrading RNA.
  • RNA enzyme is RNA enzyme A.
  • RNA enzyme A is a single-chain polypeptide containing 4 disulfide bonds, with a molecular weight of about 13.7kDa. As an endoribonuclease, it specifically degrades cytosine (C) or uracil (U) residues on single-stranded RNA. More preferably, the concentration of RNA enzyme A is 8-12mg/ml (storage concentration). RNA enzyme A needs to be diluted when used, and the concentration of the diluted RNA enzyme A is 0.01-0.05mg/ml (working concentration).
  • the "third pH stabilizer” is a reagent used to maintain the pH value of the elution buffer within a certain range (e.g., pH 7.0-8.5).
  • the third pH stabilizer can be a pH stabilizer commonly used in the art, including: a Tris-HCl buffer pair, a buffer pair of Na2HPO4 and KH2PO4 , a buffer pair of NaH2PO4 and Na2HPO4 , or a buffer pair of NH4Cl and NH3 ⁇ H2O , etc.
  • the third pH stabilizer is a Tris-HCl buffer pair.
  • the present invention also provides a method for extracting DNA from a plant tissue sample, comprising the following steps:
  • the protease was used at a concentration of 0.2-0.6 mg/ml (working concentration).
  • the step of lysing the cell membrane further comprises using a cell
  • the cell filter is used for filtration, and the filter diameter of the cell filter is 20 ⁇ m-120 ⁇ m.
  • the mass ratio of the plant tissue sample to the total volume of the lysis buffer A and the lysis buffer B is 1:(2-70), in g/mL. More preferably, the mass ratio of the plant tissue sample to the total volume of the lysis buffer A and the lysis buffer B is 1:(5-50), in g/mL.
  • the present invention uses a larger volume of lysis and digestion system to ensure sufficient lysis of the cell nucleus.
  • the step of lysing the cell nucleus comprises: first lysing at 30-39° C. for 10-40 minutes, and then lysing at 45-55° C. for 2-3 hours.
  • the present invention adopts a lower lysis temperature, which can prevent serious breakage of DNA during the digestion process, and adopts a longer lysis time, so that the lysis and digestion treatment is more complete.
  • the step of purifying the released DNA comprises:
  • the organic solvent includes phenol, chloroform or isoamyl alcohol.
  • An equal volume of phenol; a mixed solution of phenol, chloroform and isoamyl alcohol; a mixed solution of chloroform and isoamyl alcohol can be used for extraction once each, for example, first extract with phenol, then add a mixed solution of phenol, chloroform and isoamyl alcohol (volume ratio 25:24:1), and finally add a mixed solution of chloroform and isoamyl alcohol (volume ratio 24:1).
  • the alcohol solution is a 70-80% ethanol solution.
  • the extraction is performed at a rotation speed of 10-20rpm. Using a low-speed horizontal shaker during the extraction process can minimize the damage to the DNA molecules caused by the extraction.
  • the volume of the ethanol solution is 1.5-2 times the volume of the extraction solution containing DNA to reduce the precipitation of low molecular weight DNA.
  • the step of purifying the released DNA further comprises:
  • Dissolving DNA at the above temperature can accelerate the dissolution of long-fragment DNA in the elution buffer to obtain a pure DNA solution.
  • Cell nucleus separation Add washing buffer C to the leaf tissue, use a tissue homogenizer to break the cells in a homogenized manner, and filter and wash to obtain a cell nucleus precipitate. By separating the cell nucleus, impurities outside the cell nucleus are removed as much as possible to reduce the contamination of impurities in the cell during subsequent DNA extraction.
  • Lysis buffer A and lysis buffer B are added to the nuclear pellet to lyse the nuclei and release the DNA, which is then digested with proteinase K and RNase A to degrade most impurities such as proteins and RNA.
  • the cell nucleus separation step is added, in which the present invention specifically lyses the cell membrane structure, releases the intact cell nucleus, reduces the contamination of cell impurities, and helps to improve the efficiency of lysis and digestion; in the cell nucleus lysis step, the present invention uses a lower digestion temperature and a mild SDS lysis solution to prevent serious breakage of DNA during the digestion process; in the DNA purification step, the present invention uses a low-speed horizontal shaker during the extraction process to minimize the damage of the extraction to the DNA molecules.
  • the following examples compare the extraction method of the present invention with the method of extracting DNA using the Circulomics plant DNA extraction kit, and it can be found that: the purity of the DNA prepared by the present invention is much higher than the purity of the DNA extracted using the Circulomics plant DNA extraction kit, while the length of the DNA is comparable.
  • the present invention uses a cleaning buffer C containing a combination of EDTA and EGTA, which more effectively inhibits the activity of DNA enzymes and ensures the integrity of the cell nucleus; in the cell nucleus lysis step, the lysis and digestion system of the present invention is larger, the reaction time is longer, and the lysis and digestion treatment is more complete; in the DNA purification step, the present invention performs organic solvent extraction after lysis and digestion, which removes the contamination of organic impurities to the greatest extent.
  • the kit of the present invention was used to extract DNA from 0.15 g of Dieffenbachia cerasifera leaves.
  • the kit of the present invention comprises the following components:
  • Washing buffer A contains 20mM Tris-HCl, 50mM EDTA, 50mM EGTA, 160mM KCl, 1M sucrose, 1% (v/v) Triton X-100 and 2% (w/v) PVP, pH 8.0;
  • Washing buffer B contains 100 mM spermine and 100 mM spermidine; before the experiment, washing buffer B is prepared by spermine and spermidine with sterile water;
  • Lysis buffer B contains 20% (w/v) SDS, the mass volume ratio of SDS to water is 20g:100ml; Lysis buffer B needs to be diluted when used, and the concentration of SDS after dilution is 1.25%;
  • RNase A 10mg/ml RNase A; RNase A needs to be diluted when used, and the concentration of RNase A after dilution is 0.03mg/ml;
  • proteinase K needs to be diluted when used, and the concentration of proteinase K after dilution is 0.4mg/ml;
  • Elution buffer contains 10 mM Tris-HCl, pH 8.0;
  • This embodiment mainly includes four steps of cell nucleus separation, cell nucleus lysis, organic solvent extraction, DNA precipitation and dissolution (as shown in FIG. 4 ), and the specific steps are as follows:
  • washing buffer A 1 ml of washing buffer A to the crude cell nucleus pellet, use a brush to completely disperse the cell nuclei, add washing buffer C to 40 ml, and invert to mix.
  • Step 1.8 can be repeated several times until there is no obvious precipitation at the bottom of the centrifuge tube after centrifugation.
  • washing buffer A 1 ml of washing buffer A to the crude cell nucleus pellet, use a brush to completely disperse the cell nuclei, add washing buffer C to 40 ml, and invert to mix.
  • step 1.14 Take out the cell nuclear solution obtained in step 1.14, centrifuge at 3500 rcf and 4°C for 15 min, completely remove the supernatant, and obtain the cell nuclear precipitate.
  • the kit of the present invention was used to extract DNA from 0.15 g of Dieffenbachia cerasifera leaves.
  • Elution buffer contains 10 mM Tris-HCl, pH 8.0;
  • Washing buffer A contains 10mM Tris-HCl, 50mM EDTA, 50mM EGTA, 160mM KCl, 1M sucrose, 1% (v/v) Triton X-100 and 2% (w/v) PVP, pH 8.0;
  • Lysis buffer A contains 10mM Tris-HCl, 25mM EDTA and 100mM NaCl, pH 8.0;
  • proteinase K needs to be diluted when used, and the concentration of proteinase K after dilution is 0.4mg/ml;
  • Washing buffer A contains 200 mM Tris-HCl, 50 mM EDTA, 50 mM EGTA, 160 mM KCl, 1 M sucrose, 1% (v/v) Triton X-100 and 2% (w/v) PVP, pH 8.0;
  • the kit of the present invention was used to extract DNA from 0.15 g of Dieffenbachia cerasifera leaves.
  • RNase A 10mg/ml RNase A; RNase A needs to be diluted when used, and the concentration of RNase A after dilution is 0.03mg/ml;
  • washing buffer A contained: 10mM Tris-HCl, 5mM EDTA, 25mM EGTA, 80mM potassium chloride, 0.5M sucrose, 0.5% (v/v) Triton X-100, 1% (w/v) PVP, 1mM spermine, 1mM spermidine and 0.2% (v/v) ⁇ -mercaptoethanol.
  • the kit of the present invention comprises the following components:
  • Washing buffer A contains 20mM Tris-HCl, 140mM EDTA, 50mM EGTA, 160mM KCl, 1M sucrose, 1% (v/v) Triton X-100 and 2% (w/v) PVP, pH 8.0;
  • Washing buffer B contains 100 mM spermine and 100 mM spermidine; before the experiment, washing buffer B is prepared by spermine and spermidine with sterile water;
  • Lysis buffer A contains 10mM Tris-HCl, 25mM EDTA and 100mM NaCl, pH 8.0;
  • Lysis buffer B contains 20% (w/v) SDS, the mass volume ratio of SDS to water is 20g:100ml; Lysis buffer B needs to be diluted when used, and the concentration of SDS after dilution is 1.25%;
  • RNase A 10mg/ml RNase A; RNase A needs to be diluted when used, and the concentration of RNase A after dilution is 0.03mg/ml;
  • proteinase K needs to be diluted when used.
  • concentration of proteinase K was 0.4 mg/ml;
  • Elution buffer contains 10 mM Tris-HCl, pH 8.0;
  • washing buffer A contained: 10mM Tris-HCl, 70mM EDTA, 25mM EGTA, 80mM potassium chloride, 0.5M sucrose, 0.5% (v/v) Triton X-100, 1% (w/v) PVP, 1mM spermine, 1mM spermidine and 0.2% (v/v) ⁇ -mercaptoethanol.
  • the kit of the present invention was used to extract DNA from 0.15 g of Dieffenbachia cerasifera leaves.
  • the kit of the present invention comprises the following components:
  • Washing buffer A contains 20mM Tris-HCl, 50mM EDTA, 10mM EGTA, 160mM KCl, 1M sucrose, 1% (v/v) Triton X-100 and 2% (w/v) PVP, pH 8.0;
  • Washing buffer B contains 100 mM spermine and 100 mM spermidine; before the experiment, washing buffer B is prepared by spermine and spermidine with sterile water;
  • Lysis buffer A contains 10 mM Tris-HCl, 25 mM EDTA, and 100 mM NaCl, pH 8.0;
  • Lysis buffer B contains 20% (w/v) SDS, the mass volume ratio of SDS to water is 20g:100ml; Lysis buffer B needs to be diluted when used, and the concentration of SDS after dilution is 1.25%;
  • RNase A 10mg/ml RNase A; RNase A needs to be diluted when used, and the concentration of RNase A after dilution is 0.03mg/ml;
  • proteinase K needs to be diluted when used, and the concentration of proteinase K after dilution is 0.4mg/ml;
  • Elution buffer contains 10 mM Tris-HCl, pH 8.0;
  • the kit of the present invention was used to extract DNA from 0.15 g of Dieffenbachia cerasifera leaves.
  • the kit of the present invention comprises the following components:
  • Washing buffer A contains 20mM Tris-HCl, 50mM EDTA, 140mM EGTA, 160mM KCl, 1M sucrose, 1% (v/v) Triton X-100 and 2% (w/v) PVP, pH 8.0;
  • Washing buffer B contains 100 mM spermine and 100 mM spermidine; before the experiment, washing buffer B is prepared by spermine and spermidine with sterile water;
  • Lysis buffer A contains 10mM Tris-HCl, 25mM EDTA and 100mM NaCl, pH 8.0;
  • RNase A 10mg/ml RNase A; RNase A needs to be diluted when used, and the concentration of RNase A after dilution is 0.03mg/ml;
  • proteinase K needs to be diluted when used, and the concentration of proteinase K after dilution is 0.4mg/ml;
  • Elution buffer contains 10 mM Tris-HCl, pH 8.0;
  • wash buffer A contained: 10mM Tris-HCl, 25mM EDTA, 70mM EGTA, 80mM potassium chloride, 0.5M sucrose, 0.5% (v/v) Triton X-100, 1% (w/v) PVP, 1 mM spermine, 1 mM spermidine, and 0.2% (v/v) ⁇ -mercaptoethanol.
  • the kit of the present invention was used to extract DNA from 0.15 g of Dieffenbachia cerasifera leaves.
  • the kit of the present invention comprises the following components:
  • Washing buffer B contains 100 mM spermine and 100 mM spermidine; before the experiment, washing buffer B is prepared by spermine and spermidine with sterile water;
  • RNase A 10mg/ml RNase A; RNase A needs to be diluted when used, and the concentration of RNase A after dilution is 0.03mg/ml;
  • proteinase K needs to be diluted when used, and the concentration of proteinase K after dilution is 0.4mg/ml;
  • Elution buffer contains 10 mM Tris-HCl, pH 8.0;
  • proteinase K needs to be diluted when used, and the concentration of proteinase K after dilution is 0.4mg/ml;
  • Elution buffer contains 10 mM Tris-HCl, pH 8.0;
  • RNase A 10mg/ml RNase A; RNase A needs to be diluted when used, and the concentration of RNase A after dilution is 0.03mg/ml;
  • Lysis buffer B contains 20% (w/v) SDS, the mass volume ratio of SDS to water is 20g:100ml; Lysis buffer B needs to be diluted when used, and the concentration of SDS after dilution is 1.25%;
  • Washing buffer B contains 100 mM spermine and 100 mM spermidine; before the experiment, washing buffer B is prepared by spermine and spermidine with sterile water;
  • Lysis buffer A contains 10mM Tris-HCl, 25mM EDTA and 100mM NaCl, pH 8.0;
  • washing buffer A contained: 10mM Tris-HCl, 25mM EDTA, 25mM EGTA, 80mM potassium chloride, 0.5M sucrose, 0.5% (v/v) Triton X-100, 1% (w/v) PVP, 1mM spermine, 1mM spermidine and 0.2% (v/v) ⁇ -mercaptoethanol.
  • Washing buffer A contains 20mM Tris-HCl, 50mM EDTA, 50mM EGTA, 160mM KCl, 1M sucrose, 1% (v/v) Triton X-100 and 2% (w/v) PVP, pH 8.0;
  • Lysis buffer A contains 10mM Tris-HCl, 25mM EDTA and 100mM NaCl, pH 8.0;
  • Lysis buffer B contains 20% (w/v) SDS, the mass volume ratio of SDS to water is 20g:100ml; Lysis buffer B needs to be diluted when used, and the concentration of SDS after dilution is 1.25%;
  • RNase A 10mg/ml RNase A; RNase A needs to be diluted when used, and the concentration of RNase A after dilution is 0.03mg/ml;
  • proteinase K needs to be diluted when used, and the concentration of proteinase K after dilution is 0.4mg/ml;
  • Elution buffer contains 10 mM Tris-HCl, pH 8.0;
  • the kit of the present invention was used to extract DNA from 0.8 g of potato leaves.
  • the kit of the present invention comprises the following components:
  • Washing buffer A contains 20mM Tris-HCl, 50mM EDTA, 50mM EGTA, 160mM KCl, 1M sucrose, 1% (v/v) Triton X-100 and 2% (w/v) PVP, pH 8.0;
  • Washing buffer B contains 100 mM spermine and 100 mM spermidine; before the experiment, washing buffer B is prepared by spermine and spermidine with sterile water;
  • Lysis buffer B contains 20% (w/v) SDS, the mass volume ratio of SDS to water is 20g:100ml; Lysis buffer B needs to be diluted when used, and the concentration of SDS after dilution is 1.25%;
  • RNase A 10mg/ml RNase A; RNase A needs to be diluted when used, and the concentration of RNase A after dilution is 0.03mg/ml;
  • proteinase K needs to be diluted when used, and the concentration of proteinase K after dilution is 0.4mg/ml;
  • Elution buffer contains 10 mM Tris-HCl, pH 8.0;
  • wash buffer A contained: 10mM Tris-HCl, 25mM EDTA, 25mM EGTA, 80mM potassium chloride, 0.5M sucrose, 0.5% (v/v) Triton X-100, 1% (w/v) PVP, 1 mM spermine, 1 mM spermidine, and 0.2% (v/v) ⁇ -mercaptoethanol.
  • the kit of the present invention was used to extract DNA from 1 g of cabbage leaves.
  • the kit of the present invention comprises the following components:
  • Washing buffer B contains 100 mM spermine and 100 mM spermidine; before the experiment, washing buffer B is prepared by spermine and spermidine with sterile water;
  • Lysis buffer A contains 10mM Tris-HCl, 25mM EDTA and 100mM NaCl, pH 8.0;
  • Lysis buffer B contains 20% (w/v) SDS, the mass volume ratio of SDS to water is 20g:100ml; Lysis buffer B needs to be diluted when used, and the concentration of SDS after dilution is 1.25%;
  • proteinase K needs to be diluted when used, and the concentration of proteinase K after dilution is 0.4mg/ml;
  • Elution buffer contains 10 mM Tris-HCl, pH 8.0;
  • washing buffer A contained: 10mM Tris-HCl, 25mM EDTA, 25mM EGTA, 80mM potassium chloride, 0.5M sucrose, 0.5% (v/v) Triton X-100, 1% (w/v) PVP, 1mM spermine, 1mM spermidine and 0.2% (v/v) ⁇ -mercaptoethanol.
  • the cleaning buffer C in Example 19 was replaced with a traditional NIB buffer, which contained: 10 mM MES-KOH (pH 5.4), 10 mM NaCl, 10 mM potassium chloride, 2.5 mM EDTA, 0.25 M sucrose, 1% (w/v) PVP-40, 0.1 mM spermine, 0.5 mM spermidine, 1 mM DTT, and 0.5% (v/v) Triton X-100.
  • a traditional NIB buffer which contained: 10 mM MES-KOH (pH 5.4), 10 mM NaCl, 10 mM potassium chloride, 2.5 mM EDTA, 0.25 M sucrose, 1% (w/v) PVP-40, 0.1 mM spermine, 0.5 mM spermidine, 1 mM DTT, and 0.5% (v/v) Triton X-100.
  • the remaining materials and steps were the same as in Example 19.
  • the cleaning buffer C in Example 19 is replaced by a cleaning buffer D, which contains: 10 mM Tris-HCl, 25 mM EDTA, 80 mM potassium chloride, 0.5 M sucrose, 0.5% (v/v) Triton X-100, 1% (w/v) PVP-40, 1 mM spermine, 1 mM spermidine and 0.2% (v/v) ⁇ -mercaptoethanol, but does not contain EGTA.
  • the remaining materials and steps are the same as in Example 19.
  • This comparative example adopts the common CTAB method to extract DNA from corn leaves, and the specific steps are as follows.
  • CTAB lysis buffer contains: 100 mM Tris-HCl, 50 mM EDTA, 1 M NaCl, 1% (w/v) PVP-40, 0.6 M LiCl, 4 M urea, and 2% (w/v) CTAB.
  • This comparative example uses the Circulomics plant DNA extraction kit to extract wheat leaf samples, and the specific steps are as follows:
  • step 2.18 Dissolve the solution obtained in step 2.16 at room temperature overnight to obtain the DNA extraction solution.
  • the 260/280 of the present invention and the DNA extracted by the conventional CTAB method are both about 1.8, and the 260/230 are both about 2.0, and the DNA purity meets the sequencing requirements of the ONT platform.
  • the sample DNA to be tested was added to the prepared 0.7% agarose gel containing nucleic acid dye, with ⁇ DNA/HindIII (Tiangen, MD202) and Lambda PFG Ladder (NEB, N0341S), the electrophoresis buffer was 0.5XTBE, the pulse field electrophoresis conditions were voltage 6.5V/cm, pulse conversion time 1-20s, and electrophoresis time 16-20 hours. After the electrophoresis, the results were photographed and recorded with a gel imager.
  • the length of DNA extracted in Examples 1-20 in Figures 1 and 2 is about 23.1-339.5 kb, which is relatively long. That is, when the concentration of each component in the washing buffer A and the lysis buffer A is changed within a certain range, the length of the extracted DNA is relatively long and the extraction effect is relatively good.
  • Comparison of Example 19 and Comparative Example 1 in Figures 2 and 3 shows that for wheat samples, the length of DNA obtained after washing with the washing buffer C of the present invention is between 23.1 and 339.5 kb, while the length of DNA obtained after washing with the traditional NIB washing buffer is between 9.4 and 23.1 kb.
  • the washing buffer C of the present invention is far superior to the traditional NIB washing buffer.
  • Example 19 By comparing Example 19 and Comparative Example 2 in Figures 2 and 3, it can be seen that for the same wheat sample, the length of the DNA obtained after washing with the washing buffer C of the present invention is between 23.1-339.5 kb, while the length of the DNA obtained after washing with the washing buffer D without EGTA is between 23.1-97 kb, indicating that EGTA in the washing buffer C of the present invention plays a crucial role in extracting ultra-long fragment DNA.
  • Comparison of Example 20 and Comparative Example 3 in Figures 2 and 3 shows that, for the same corn sample, the length of DNA extracted by the present invention is between 23.1-436.5 kb, while the length of DNA extracted by the conventional CTAB method is about 23.1-97 kb.
  • the length of DNA prepared by the present invention is much better than that by the conventional CTAB method.
  • Comparison of Example 19 and Comparative Example 4 in FIG. 2 and FIG. 3 shows that, for both wheat samples, the length of DNA extracted by the present invention is between 23.1-339.5 kb, while the length of DNA extracted by the circulomics plant DNA extraction kit is also between 23.1-339.5 kb, but it has a brighter band at 23.1-97 kb, that is, most of the DNA is between 23.1-97 kb, and the DNA is still short.
  • the length of DNA prepared by the present invention is better than that of the circulomics plant DNA extraction kit.

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Abstract

La présente invention concerne un kit et un procédé d'extraction d'ADN dans un échantillon de tissu végétal. Le kit comprend une solution tampon de lavage A, une solution tampon de lavage B, une solution tampon de lyse A, une solution tampon de lyse B et une protéase. La solution tampon de lavage A comprend entre 10 et 300 mM d'un premier stabilisateur de pH, entre 10 et 400 mM d'un premier chélateur d'ions métalliques, entre 10 et 2300 mM d'un stabilisateur de pression osmotique, entre 0,4 et 4 % (v/v) d'un tensioactif non ionique et entre 1 et 10 % (p/v) d'un premier antioxydant, et la valeur pH de la solution tampon de lavage A est comprise entre 7,0 et 8,5 ; la solution tampon de lavage B comprend entre 10 et 400 mM d'un premier stabilisateur d'ADN ; la solution tampon de lyse A comprend entre 1 et 200 mM d'un deuxième stabilisateur de pH, entre 1 et 200 mM d'un deuxième agent chélateur d'ions métalliques et entre 10 et 300 mM d'un deuxième stabilisateur d'ADN, et le pH de la solution tampon de lyse A est compris entre 7,0 et 8,5 ; la solution tampon de lyse B comprend entre 10 et 30 % (p/v) d'un agent tensioactif anionique ; et la concentration de la protéase est comprise entre 1 et 30 mg/ml.
PCT/CN2023/124174 2022-12-26 2023-10-12 Kit et procédé d'extraction d'adn dans un échantillon de tissu végétal WO2024139530A1 (fr)

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Application Number Priority Date Filing Date Title
CN202211673426.4A CN118291441A (zh) 2022-12-26 2022-12-26 一种用于提取植物组织样本中dna的试剂盒及方法
CN202211673426.4 2022-12-26

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