CN113862258A - High-yield DNA fragment and preparation method thereof - Google Patents
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Abstract
The invention belongs to the biotechnology, and particularly relates to a high-yield DNA fragment and a preparation method thereof. The invention prepares the fragmented DNA by combining the nucleosome enzyme cutting method with column purification, the process of the obtained DNA fragment is similar to the process of the DNA fragment naturally generated in a human body, and the size distribution structure of the DNA is more consistent than that of the DNA fragment obtained by ultrasonic breaking.
Description
Technical Field
The invention belongs to the biotechnology, and particularly relates to a DNA fragment and a preparation method thereof.
Background
The tumors seriously threaten the health and survival of human beings, and according to a 2018 global cancer statistical data report issued on line by a clinician journal of cancer in the United states, about 1810 ten thousand new cancer cases and 960 ten thousand cancer death cases are published all year round. Therefore, early diagnosis is crucial to improve patient prognosis. The mutation of various genes exists in the process of generating tumors, and the detection of the mutation types existing in patients not only helps to understand the generating mechanism of the tumors, but also can be used for the diagnosis and prognosis evaluation of the tumors. At present, in clinical, fresh tissues and paraffin-embedded tissues are mostly adopted for tumor gene mutation detection, so that a basis is provided for treatment of patients, but certain limitations exist: local material collection cannot reflect tumor heterogeneity, certain tumor anatomical positions are not suitable for puncture, and the puncture is difficult to dynamically monitor the progress process of the tumor.
The DNA fragment released from tumor and tumor metastasis into blood is called ctDNA (circulating tumor DNA), has a length of about 166bp, and carries tumor gene mutation information. Compared with tissue detection, the blood ctDNA gene mutation detection has the following remarkable advantages: the material is convenient to obtain, the tumor heterogeneity can be reflected, the dynamic mutation state of the tumor gene can be monitored in real time, and the patient compliance is good. According to recent research reports at home and abroad, ctDNA gene mutation detection is mainly applied to clinic: tumor diagnosis, treatment guidance, prognosis evaluation, tumor recurrence and metastasis monitoring.
Liquid biopsy (LiquidBoispsy) was rated by MITTECHNOLOGY review in 2015 as one of the ten years' scientific breakthroughs. Liquid biopsy refers to sampling and analyzing of non-solid biological tissues mainly comprising blood, is a new disease diagnosis and monitoring tool, and can be applied to a series of diseases such as cancer, heart disease, prenatal diagnosis, organ transplantation and the like. ctDNA gene mutation detection methods are roughly divided into two types, one is a detection method based on Polymerase Chain Reaction (PCR); the other method is a Next Generation Sequencing (NGS) method. On the basis of the two methods, researchers develop a plurality of gene mutation detection technologies, such as a mutation enrichment PCR technology, a multiplexing PCR technology, a high-throughput parallel sequencing and polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP) technology, a BEAMing technology, a digital PCR technology and the like. These techniques all have their own features, however standardization and normalization of ctDNA detection technology is a key issue for their clinical applications. Since the Reference Material (RM) is a scale for evaluating the level of operation of instruments, apparatuses, detection methods, and personnel, and controlling the quality of products in the production process and the inspection process, the ctDNA reference material is in urgent need of development.
At present, fragmented DNA used for preparing cfDNA standard substances, quality control substances and reference substances can be obtained by a method for artificially synthesizing plasmids, but the method is gradually eliminated because the artificially synthesized plasmids cannot simulate the sequence complexity of natural cfDNA in a normal biological state, and the method for obtaining the fragmented DNA by breaking genome DNA of a cell line by ultrasonic is a main method for obtaining the fragmented DNA, but because the process of breaking the DNA by mechanical force does not exist in a human body, DNA fragments obtained by breaking by ultrasonic have larger differences with the sizes of human natural cfDNA fragments and DNA molecules, and specifically: the size distribution of DNA fragments obtained by ultrasonic breaking is obviously different from that of natural ctDNA, the size of the natural ctDNA fragments is normally distributed within 150bp-180bp, and 166bp of a DNA fragment distribution concentration region is more than or equal to 85%; the DNA fragments obtained by ultrasonic breaking are normally distributed between 120bp and 200bp, and the distribution concentration area 160bp of the DNA fragments is more than or equal to 60 percent. The biological characteristics of the tail end of a DNA fragment obtained by ultrasonic breaking are remarkably different from those of natural ctDNA, the natural ctDNA is mainly generated by an in vivo enzymolysis process, the tail end of a DNA molecule is 5 'end phosphate and 3' end hydroxyl, and the conditions of bases of 5 'and 3' protruding ends, 5 'hydroxyl and 3' end phosphate can occur in the fragmented DNA obtained by ultrasonic breaking; therefore, in the case of single strand sequencing and library building of NGS, ultrasonic disruption of the obtained fragmented DNA must be end-repaired, i.e. the bases of the 5 'and 3' overhangs are removed, while ensuring that the 5 'end is phosphate and the 3' end is hydroxyl for a addition and library building.
Disclosure of Invention
The process of the obtained DNA fragment is similar to that of the DNA fragment naturally generated in a human body, and the size distribution structure of the DNA is more consistent with that of the DNA fragment naturally generated in the human body compared with that of the DNA fragment obtained by breaking with ultrasonic waves.
The invention adopts the following technical scheme:
adding the mixed solution into cells, shaking, then incubating with ice, adding a non-ionic surfactant solution, shaking, incubating with ice, then centrifuging, then discarding the supernatant, then adding a micrococcal nuclease buffer solution, blowing, then centrifuging, then discarding the supernatant, then adding a micrococcal nuclease buffer solution for resuspension, then adding micrococcal nuclease, shaking, then performing water bath treatment, then adding an ethylene diamine tetraacetic acid solution and a ribonuclease A solution, shaking, performing water bath treatment, then adding a sodium dodecyl sulfate solution and a protease K solution, shaking, performing water bath treatment, then performing purification and recovery, and obtaining the DNA fragment.
In the invention, the mixed solution comprises a 4-hydroxyethyl piperazine ethanesulfonic acid solution, an inorganic magnesium salt solution, an inorganic potassium salt solution, an organic reducing agent solution and a phenylmethylsulfonyl fluoride solution, and preferably comprises a 4-hydroxyethyl piperazine ethanesulfonic acid solution, an inorganic magnesium salt solution, an inorganic potassium salt solution, an organic reducing agent solution, a phenylmethylsulfonyl fluoride solution and water; preferably, the volume ratio of the 4-hydroxyethyl piperazine ethanesulfonic acid solution to the inorganic magnesium salt solution to the inorganic potassium salt solution to the organic reducing agent solution to the phenylmethylsulfonyl fluoride solution to water is (5-15) to (0.5-3) to (5-15) to (0.1-1) to (5-15) to (800-1200), and preferably is (8-12) to (1-2) to (8-12) to (0.3-0.8) to (8-12) to (900-1050). The concentration of the 4-hydroxyethyl piperazine ethanesulfonic acid solution is 0.08-0.12M, the pH value is 7.5-8, and the solution is a conventional reagent; the concentration of the inorganic magnesium salt solution is 0.8-1.2M, the concentration of the inorganic sylvite solution is 0.8-1.2M, the concentration of the organic reducing agent solution is 0.8-1.2M, and the concentration of the phenylmethylsulfonyl fluoride solution is 0.08-0.12M. Wherein the inorganic magnesium salt comprises magnesium chloride, the inorganic potassium salt comprises potassium chloride, and the organic reducing agent comprises dithiothreitol DTT.
In the present invention, the number of cells is 1X 107～1×109And (4) respectively. The nonionic surfactant is CA-630.
In the invention, the volume ratio of the mixed solution, the nonionic surfactant solution, the ethylene diamine tetraacetic acid solution, the ribonuclease A solution, the sodium dodecyl sulfate solution and the protease K solution is 1000 to (5-15) to (30-50) to (3-18) to (70-150) to (10-30), preferably 1000 to (8-12) to (35-45) to (8-13) to (90-110) to (15-25).
In the invention, the using amount of the micrococcus nuclease buffer solution is 0.8-1.2 times, preferably 0.9-1.1 times of the volume of the mixed solution; the volume of the buffer solution for the two uses of the micrococcal nuclease may be the same or different. The composition of the micrococcal nuclease buffer is exemplified as follows: 50 mM Tris-HCl (pH = 8.0), 5 mM CaCl20.1 mg/mL BSA (bovine serum albumin), and water.
In the invention, specific operations of shaking, ice incubation, water bath, centrifugation and whipping are conventional techniques, wherein the shaking time is not particularly limited, and the purpose is conventional and is to mix uniformly; the temperature of the centrifugal treatment is 0-5 ℃, the centrifugal force is 700-1000 g, and the time is 3-10 minutes; the ice incubation time is 3-15 minutes; the blow beating is carried out for 2-5 times by a liquid transfer device; the temperature of the water bath treatment is 35-60 ℃, and the time is 5-45 minutes.
In the invention, an adsorption column is adopted for purification and recovery to obtain DNA fragments, and the specific operation is carried out according to the instruction.
The invention prepares fragmented DNA by combining nucleosome enzyme cutting method with column purification, the process of the obtained DNA fragment is similar to the process of the DNA fragment naturally generated in human body, the size distribution structure of the DNA is more consistent than that of the DNA fragment obtained by ultrasonic breaking, and the biological characteristics of the tail end of the obtained DNA fragment are similar to those of natural ctDNA: the fragmented DNA generated in a human body is mainly generated by an in vivo enzymolysis process, the tail ends of DNA molecules are 5 'end phosphoric acid and 3' end hydroxyl, the tail ends of the fragmented DNA molecules obtained by the invention are 5 'end phosphoric acid and 3' end hydroxyl, when NGS detection is performed to build a library, the connection efficiency of natural ctDNA molecules is 61%, and the connection efficiency of the fragmented DNA molecules obtained by the invention is 65%; the complexity of the DNA fragment obtained by the invention is similar to that of a natural ctDNA sequence, the size distribution of the DNA fragment is highly consistent with that of the natural ctDNA, and the mutation is randomly distributed on the position of the DNA fragment, so that the sensitivity and the accuracy of the detection equipment and the detection method can be reflected and evaluated more truly.
Drawings
Fig. 1 is a size distribution graph of natural cfDNA fragments;
FIG. 2 is a graph showing the size distribution of DNA fragments prepared in the first example;
FIG. 3 is a size distribution diagram of a DNA fragment prepared in comparative example one;
FIG. 4 is a size distribution diagram of a DNA fragment prepared in comparative example two.
Detailed Description
The raw materials adopted by the invention are conventional in the field, the pretreatment of the cells is also conventional in the field, and the specific preparation operation method and the test method are conventional in the field.
Purchasing a target cell line from a cell bank, downloading a cell culture instruction on a cell bank website, and preparing a complete culture medium suitable for cell growth for later use; the purchased cell line arrives at a cell culture room in a freezing mode, the cells are put into a water bath kettle at 37 ℃ for fast thawing, meanwhile, the freezing tube is gently shaken continuously, after the cell suspension in the freezing tube is melted, the freezing tube is sprayed with 75% alcohol for disinfection, the freezing tube is moved into a biological safety cabinet, the freezing tube is opened, the cell suspension is sucked out and added into 5ml of complete culture medium, the cell suspension is moved into a centrifuge again, the centrifuge is centrifuged at 1000rpm for 8min, after the centrifugation is finished, the supernatant is removed, 1ml of complete culture medium is used for uniformly mixing tube bottom sediment, the cell number and the cell activity are calculated after the sampling and trypan blue are mixed by 1:1 volume, the complete culture medium is added to adjust the cell density to 1 × 10 according to the counting result6mL, for transfer to cell culture 6-well plates, 2 mL/well, cell culture plates were transferred to 37 ℃ with 5% CO2Culturing in an incubator with 95% humidity; sampling every 48 hours, counting the number of cells, adding complete culture medium to maintain the cell density at 0.5-1 × 106and/mL. The cell amount is larger than 1 × 108Collect cells into 1.5mL centrifuge tubes, 1X 108Tube for storage at-20 ℃ while collecting a tube of cells at a cell count of 1X 106And performing STR identification of the cells. Comparing the obtained STR typing result with professional STR database, confirming that the purchased cell line is correct and is not polluted by other cells, and performing cell analysisThe cell matching degree of the cell is more than or equal to 80 percent according to ANSI standard.
The method for preparing the DNA fragment disclosed by the invention comprises the following steps:
taking out the cells from a refrigerator at-20 deg.C, placing on ice for 20min, and thawing; 1X 108To the cells were added 10. mu.L of 0.1M Hepes (4-hydroxyethylpiperazine ethanesulfonic acid) aqueous solution and 1.5. mu.L of 1M MgCl2Aqueous solution, 10. mu.L of 1M KCl aqueous solution, 0.5. mu.L of 1M DTT aqueous solution, 10. mu.L of 0.1M PMSF (phenylmethylsulfonyl fluoride) aqueous solution and 970. mu.L of water, shaking for 15s, and incubating for 10min on ice; then adding 10uL 10% IGEPAL CA-630 aqueous solution, vibrating for 10s, and incubating for 5min with ice; centrifuging at 4 deg.C and 900g for 5min, and removing supernatant; then adding 1000 mu L micrococcal nuclease buffer solution for cleaning, blowing and beating for 3 times by a pipette at 4 ℃ and 900g, centrifuging for 5min, removing supernatant, adding 1000 mu L micrococcal nuclease buffer solution for resuspending cell nucleus, adding 100uL micrococcal nuclease (100U/uL), shaking and uniformly mixing, and carrying out water bath at 42 ℃ for 10 min; then adding 40uL EDTA (Thermo R1021), shaking and mixing uniformly; adding 10uL of RNaseA (ribonuclease A) Tris-HCL buffer solution (Tris (hydroxymethyl) aminomethane hydrochloride buffer solution) with the concentration of 20mg/mL, and shaking and mixing uniformly; water bath at 37 deg.C for 30 min; then 100uL of 10% SDS (sodium dodecyl sulfate) aqueous solution is added, and the mixture is shaken and mixed evenly; then 20uL of protease K (Proteinase K, 200 mg/mL) is added, shaken and mixed evenly; water bath at 56 deg.C for 30 min; and finally, purifying and recovering the DNA fragment to obtain fragmented DNA, wherein the micrococcus nuclease buffer solution comprises the following components: 50 mM Tris-HCl (pH = 8.0), 5 mM CaCl20.1 mg/mL BSA and water.
The present invention uses QIAQuick PCR purification Kit to purify and recover the above digested DNA fragment according to the instructions, as follows:
the samples were transferred to a 15mL centrifuge tube, and Buffer PB of 5 sample volumes in the QIAQuick PCR purification Kit, i.e., V sample: VBuffer PB = 1: 5, shaking and uniformly mixing;
taking 10 adsorption columns and collection tubes provided by QIAQuick PCR purification Kit (cat # 28106), and transferring the mixed samples to 10 adsorption columns on average;
centrifuging each adsorption column and the collection tube at 14000rpm for 30s, discarding the liquid in the collection tube, and repeating the steps until the sample is transferred;
adding 750 mu L of Buffer PE into each adsorption column, 14000rpm, centrifuging for 30s, and discarding liquid in the collection tube;
centrifuging each adsorption column at 14000rpm for 2min, and placing the adsorption column in a new EP tube;
adding 55 μ L Buffer EB into each adsorption column, keeping for 3min, centrifuging at 14000rpm for 1 min; obtaining a product DNA fragment.
The fragmented DNA obtained above was quantitatively detected using the Qubit dsDNA HS Assay kit (cat # Q32854) according to the instructions.
Example one
Using NCI-H1975 cells as an extraction sample, obtaining a fragmented DNA solution according to the method, using a Qubit dsDNA HS Assay Kits (cat No. Q32854) according to the instruction to carry out quantitative detection on the obtained fragmented DNA, and determining that the concentration of the fragmented DNA solution is 42.7 ng/muL, namely the total amount of the obtained DNA is as follows: 23485 ng.
Analysis of the size of the obtained DNA fragments using agilent 2100 Bioanalyzer the DNA fragment size distribution was similar to that of natural cfDNA, see fig. 1, fig. 2.
Example two
On the basis of the first embodiment, the column purification is replaced by magnetic bead purification, and the rest is unchanged, namely, the QIAQuick PCR purification Kit column purification is replaced by Beckmann magnetic bead purification, the specific operation is a conventional technology, and the specific purification steps are as follows:
equilibrating Beckmann magnetic beads (cat # A63882-450 mL) at room temperature for 30 min;
transferring a sample subjected to water bath at 56 ℃ for 30min into a centrifuge tube, adding 0.75mL of 10mM TRIZMA hydrochloride buffer solution, uniformly mixing by shaking, performing flash separation, adding 1.6mL of purified magnetic beads, uniformly mixing by shaking, performing flash separation, and standing at room temperature for 20 min;
transferring the sample tube to a magnetic frame, standing for 30mins, transferring the supernatant to a new centrifugal tube after the supernatant is clarified, and discarding the magnetic beads without touching the magnetic beads;
adding 0.8mL of purified magnetic beads into the supernatant, shaking, uniformly mixing, instantly separating, and standing at room temperature for 15 mins;
transferring the sample tube to a magnetic frame, standing for 40mins, clarifying the supernatant, and discarding the supernatant;
adding 2mL of 80% ethanol for cleaning, transferring the sample tube to a magnetic frame, standing for 30min, removing the supernatant after the supernatant becomes clear, and repeating the steps once again;
adding 550 mu L10 mM TRIZMA hydrochloride buffer solution for elution, shaking, mixing uniformly, instantly separating, and standing at room temperature for 20 min;
placing the sample tube on a magnetic frame, standing for 5min, and transferring the eluate to a new EP tube;
placing the EP tube on a magnetic frame, standing for 10min, and transferring the supernatant to a new EP tube;
the obtained fragmented DNA was quantitatively detected using the Qubit dsDNA HS Assay kit (cat # Q32854) according to the instructions, and the concentration of the fragmented DNA solution was found to be 17.6 ng/. mu.L, i.e., the total amount of the obtained DNA was: 9680 ng.
Comparative example 1
NCI-H1975 cells are taken as an extracted sample, the operation steps of the conventional ultrasonic fragmentation method (the main steps are according to the instructions of ZYMO Quick-DNA Midiprep Plus Kit genomic DNA extraction Kit (cat # D4075) to extract cell genomic DNA and break by using an ultrasonic break instrument Covris-S220) are adopted to obtain a fragmented DNA solution, the size of the obtained DNA fragment is analyzed by using an Agilent 2100 Bioanalyzer, and the difference between the size distribution of the DNA fragment and the natural cfDNA is large, as shown in FIG. 3.
Comparative example No. two
On the basis of the first example, the micrococcus nuclease buffer washing and resuspension were replaced with the same amount of 50 mM Tris-HCl (pH = 8.0)/water for washing and resuspension, and the remainder was unchanged, and fragmented DNA was detected to be impure by the Agilent 2100 bioanalyzer, leaving a large amount of irregular large fragment DNA, as shown in FIG. 4.
Comparative example No. three
On the basis of example two, the micrococcus nuclease buffer solution is washed and resuspended by 50 mM Tris-HCl (pH = 8.0)/water, and the rest is not changed, and the quantitative detection is carried out on the fragmented DNA obtained by the method according to the instruction by using a Qubit dsDNA HS Assay kit (cat number: Q32854), so that the total amount of the fragmented DNA is measured to be less than that of example two.
The prior art discloses some DNA fragmentation methods, although the effect that the size distribution of the DNA fragments is slightly different from that of the natural cfDNA can be achieved, the yield is low, the invention creatively provides improvement, the micrococcal nuclease buffer solution is used for cleaning and re-suspending cell nucleuses for the first time, the enzyme cutting efficiency of the micrococcal nuclease is enhanced, so that complete enzyme cutting of cell nucleusomes is achieved, the size distribution of the obtained DNA fragments is similar to that of the natural cfDNA by combining a raw material formula for processing cells and a column purification method, and the DNA yield is very high.
Claims (10)
1. A preparation method of DNA fragments is characterized by comprising the following steps of adding a mixed solution into cells, incubating with ice after shaking, adding a non-ionic surfactant solution, incubating with ice after shaking, then centrifuging, removing the supernatant, then adding a micrococcal nuclease buffer solution, blowing, centrifuging, removing the supernatant, then adding a micrococcal nuclease buffer solution for resuspension, then adding micrococcal nuclease, performing water bath treatment after shaking, then adding an ethylene diamine tetraacetic acid solution and a ribonuclease A solution, performing water bath treatment after shaking, then adding a sodium dodecyl sulfate solution and a protease K solution, performing water bath treatment after shaking, and then purifying and recycling to obtain DNA fragments; the mixed solution comprises 4-hydroxyethyl piperazine ethanesulfonic acid solution, inorganic magnesium salt solution, inorganic potassium salt solution, organic reducing agent solution and benzyl sulfonyl fluoride solution; the micrococcus nuclease buffer solution comprises Tris-HCl buffer solution and CaCl2、BSA。
2. The method of preparing a DNA fragment according to claim 1, wherein the mixed solution is composed of a 4-hydroxyethylpiperazine ethanesulfonic acid solution, an inorganic magnesium salt solution, an inorganic potassium salt solution, an organic reducing agent solution, a phenylmethylsulfonyl fluoride solution, and water.
3. The method for producing a DNA fragment according to claim 2, wherein the volume ratio of the 4-hydroxyethylpiperazine ethanesulfonic acid solution, the inorganic magnesium salt solution, the inorganic potassium salt solution, the organic reducing agent solution, the phenylmethylsulfonyl fluoride solution and water is (5-15) to (0.5-3) to (5-15) to (0.1-1) to (5-15) to (800-1200).
4. The method for producing a DNA fragment according to claim 2, wherein the concentration of the 4-hydroxyethylpiperazine ethanesulfonic acid solution is 0.08 to 0.12M, and the pH is 7.5 to 8; the concentration of the inorganic magnesium salt solution is 0.8-1.2M, the concentration of the inorganic sylvite solution is 0.8-1.2M, the concentration of the organic reducing agent solution is 0.8-1.2M, and the concentration of the phenylmethylsulfonyl fluoride solution is 0.08-0.12M; the nonionic surfactant is CA-630.
5. The method for producing a DNA fragment according to claim 1, wherein the volume ratio of the mixed solution, the nonionic surfactant solution, the ethylenediaminetetraacetic acid solution, the ribonuclease A solution, the sodium laurylsulfate solution and the proteinase K solution is 1000: 5-15: 30-50: 3-18: 70-150: 10-30.
6. The method for preparing DNA fragments according to claim 1, wherein the buffer solution for micrococcal nuclease is Tris-HCl buffer solution, CaCl2BSA and water.
7. The method for preparing a DNA fragment according to claim 1, wherein the temperature of the centrifugation is 0 to 5 ℃ and the centrifugal force is 700 to 1000g for 3 to 10 minutes; the ice incubation time is 3-15 minutes; the blow beating is carried out for 2-5 times by a liquid transfer device; the temperature of the water bath treatment is 35-60 ℃, and the time is 5-45 minutes.
8. The method of claim 1, wherein the DNA fragment is purified and recovered by using an adsorption column.
9. A DNA fragment prepared by the method for preparing a DNA fragment according to claim 1.
10. Use of a micrococcal nuclease buffer for the preparation of DNA fragments.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107841533A (en) * | 2017-11-13 | 2018-03-27 | 深圳先进技术研究院 | A kind of chromatin breaking method and its application |
| CN109022419A (en) * | 2018-08-21 | 2018-12-18 | 苏州安可济生物科技有限公司 | A kind of preparation method of fragmentation DNA |
| CN109055487A (en) * | 2018-08-09 | 2018-12-21 | 苏州安可济生物科技有限公司 | The preparation of the circulating tumor dissociative DNA standard items of simulating blood plasma matrix |
-
2021
- 2021-11-02 CN CN202111290764.5A patent/CN113862258A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107841533A (en) * | 2017-11-13 | 2018-03-27 | 深圳先进技术研究院 | A kind of chromatin breaking method and its application |
| CN109055487A (en) * | 2018-08-09 | 2018-12-21 | 苏州安可济生物科技有限公司 | The preparation of the circulating tumor dissociative DNA standard items of simulating blood plasma matrix |
| CN109022419A (en) * | 2018-08-21 | 2018-12-18 | 苏州安可济生物科技有限公司 | A kind of preparation method of fragmentation DNA |
Non-Patent Citations (3)
| Title |
|---|
| JESSE R. RAAB 等: "SWI/SNF remains localized to chromatin in the presence of SCHLAP1", NAT GENET., vol. 51, no. 1, pages 26 - 29, XP036927704, DOI: 10.1038/s41588-018-0272-z * |
| LIMINSHIYE: "QIAGEN凯杰PCR纯化试剂盒28106", WWW.LIMINSHIYE.COM, pages 1 * |
| THERMOFISHER: "Micrococcal Nuclease 88216", WWW.THERMOFISHER.COM, pages 1 * |
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