CN114231596A - Gene detection method based on CRISPR/dcas9 and magnetic nano material and application - Google Patents
Gene detection method based on CRISPR/dcas9 and magnetic nano material and application Download PDFInfo
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- 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
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N2310/00—Structure or type of the nucleic acid
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Abstract
The gene detection method based on the CRISPR/dcas9 and the magnetic nano material comprises the following steps: obtaining a target oncogene sequence to be detected, and selecting specific sgRNA; preparing streptavidin magnetic nano-microspheres, reacting the streptavidin magnetic nano-microspheres with SNAP-Biotin, and combining with SNAP-tag on CRISPR/dCas9 protein to form CRISPR @ magnetic nano-microspheres; and (2) incubating the prepared CRISPR @ magnetic nanospheres and the sgRNAs selected in the step (1) together, adding the incubated particles into a body fluid sample to be detected, and adding a nucleic acid dye, wherein when a target oncogene exists in the body fluid sample to be detected, the nucleic acid dye generates a fluorescence detection signal on the surface of the magnetic nanospheres, so that the target oncogene is detected. The invention adopts the method of combining CRIPSR/dcas9 technology with magnetic nano-microspheres, so that the body fluid sample is quickly separated, the sensitivity is improved, the signal is amplified, and the detection specificity is improved. The specificity of the detection on breast cancer related genes is strong, the molecular stability is good, the detection cost is low, and the problem of poor specificity in the existing detection method is solved.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to detection and application of CRISPR/dcas9 combined magnetic nano microspheres to breast cancer related genes.
HER-2/neu is an oncogene and is closely related to the occurrence and development of breast cancer. The incidence rates of HER-2/neu gene-related protein amplification and overexpression in simple hyperplasia, atypical hyperplasia and breast cancer of female breast are about 0%, 22.5% and 46.7%, wherein the primary invasive breast cancer is 22% -29%, the acne ductal carcinoma in situ is almost 100%, and the rate of ductal carcinoma in situ of breast is also large. Therefore, the HER-2/neu gene protein has important clinical value for early diagnosis of female breast cancer. By detecting the gene, early screening of breast cancer can be realized.
The CRISPR/Cas9 complex is a novel DNA targeted editing tool, consists of clustered regularly interspaced short palindromic repeats and CRISPR-associated protein mutants, and is an adaptive immune system widely existing in most bacteria and archaea. Guide RNA (sgRNA) in CRISPR/Cas system can recognize and combine exogenous nucleic acid molecule, thereby realizing the subsequent shearing and editing of related gene. The dCas9 protein is a mutant of Cas9 protein, namely, two nuclease activity regions of RuvC1 and HNH of a Cas9 endonuclease are mutated simultaneously, so that the endonuclease activity of the Cas9 protein is completely disappeared, only the capability of recognition into a genome guided by sgRNA is reserved, and target nucleic acid is captured by specific binding. By utilizing the specific sequence recognition characteristic of the CRISPR/dCas system, the method is applied to nucleic acid detection, and the detection sensitivity and specificity are obviously improved.
Biomagnetic separation is one of the most cost-effective methods in the technical field of biological separation, and has played an increasingly important role in many biological scientific studies. Different chemical groups are modified on the surface of the magnetic bead, so that the magnetic bead is effectively connected with ligands with biological affinity. When an external magnetic field is applied, the method can be used for quickly and conveniently separating the biomolecules. For example, avidin proteins or antibodies are modified onto magnetic beads to bind their high affinity target substances, such as nucleic acids, proteins, bacteria, viruses, cells, and the like. In addition, the biological magnetic separation technology is adopted, so that the defects that the centrifugation step is time-consuming and labor-consuming and the sample treatment link is complex and troublesome in the traditional separation method are overcome, and the operation of the whole experiment process is simpler and quicker.
Disclosure of Invention
The invention aims to provide a method for realizing portable and rapid hybridization and detection of breast cancer related genes based on CRISPR/dcas9 magnetic nanotechnology, which combines CRISPR/dcas9 technology with magnetic nanotechnology and rapidly and specifically detects breast cancer HER-2/neu genes so as to realize early screening and prognosis monitoring of breast cancer.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the gene detection method based on the CRISPR/dcas9 and the magnetic nano material comprises the following steps:
(1) and acquiring a target oncogene sequence to be detected, selecting specific sgRNAs according to the difference between the paired Tm temperature value and the delta G energy value, evaluating the specificity of the sgRNAs, and selecting the sgRNAs with the best specificity.
(2) And preparing streptavidin magnetic nano-microspheres, reacting the streptavidin magnetic nano-microspheres with SNAP-Biotin, and combining the streptavidin magnetic nano-microspheres with SNAP-tag on CRISPR/dCas9 protein to form CRISPR @ magnetic nano-microspheres.
(3) And (2) incubating the prepared CRISPR @ magnetic nanospheres and the sgRNAs selected in the step (1) together, adding the incubated particles into a body fluid sample to be detected, and adding a nucleic acid dye, wherein when a target oncogene exists in the body fluid sample to be detected, the nucleic acid dye generates a fluorescence detection signal on the surface of the magnetic nanospheres, so that the target oncogene is detected.
Further, the preparation method of the streptavidin magnetic nano-microsphere comprises the following steps:
preparing superparamagnetic oil-soluble magnetic nanoparticles by high-temperature oil phase reduction method, and preparing SiO by forming reversed microemulsion by using the prepared magnetic nanoparticles as magnetic cores by water-in-oil method2Magnetic nano-microsphere with core-shell structure as shell; with aminoethyl aminopropyl polydimethylsiloxaneAnd (3) pretreating the prepared magnetic nano-microspheres by using a buffer solution, and then wrapping the magnetic nano-microspheres by using streptavidin to form the streptavidin magnetic nano-microspheres.
Further, the target oncogene is HER-2/neu.
Further, the step (1) comprises:
acquiring a sequence of HER-2/neu genes, selecting specific sgRNAs according to the difference between a pairing Tm temperature value and a delta G energy value, wherein the target gene pairing Tm temperature value is 54.4 ℃, the delta G energy value is 0.33kcal/mole, selecting sgRNAs of which the pairing region Tm value with the target gene is higher than 54.4 ℃ and the delta G energy value is lower than 0.33kcal/mole, and evaluating the specificity of the selected sgRNAs to select the sgRNAs with the best specificity.
The invention also provides application of the method in preparing a breast cancer detection kit or breast cancer detection equipment.
The invention also provides a detection kit, which comprises the CRISPR @ magnetic nanospheres prepared by the invention and the selected sgRNA.
Compared with the prior art, the invention has the beneficial technical effects that:
1. the method has the advantages of strong specificity for detecting breast cancer related genes, good molecular stability and low detection cost, and solves the problem of poor specificity in the existing detection method.
2. The method of combining CRIPSR/dcas9 technology with magnetic nanometer microsphere is adopted, so that the body fluid sample is separated quickly, the sensitivity is increased, the signal is amplified, and the detection specificity is improved.
Drawings
FIG. 1 is a graph of the results of sgRNA electrophoresis of CRIPSR/dCas9 against HER-2 gene;
FIG. 2 is an electron microscope representation of magnetic nanospheres;
FIG. 3 is a schematic illustration of nucleic acid capture detection;
FIG. 4 is a graph showing the results of CRIPSR @ magnetic nanospheres combined with different HER-2 gene contents.
Detailed Description
The invention will be further illustrated in the following examples, without however being restricted thereto.
The 1 × PBS was prepared as: weighing 137mmol/LNaCl, 2.7mmol/L KCl, 10 mmol/L Na2HPO4，1.8mmol/L KH2PO4Dissolved in 800mL of water. The pH was adjusted to 7.4 and then made up to 1L.
10 × AEAPS buffer: 20mM Tris-HCl (pH 7.5), 150mM NaCl, 1mM disodium ethylenediaminetetraacetate (Na)2EDTA), 1mM ethylenediaminetetraacetic acid (EGTA), 1% Triton, 2.5mM sodium pyrophosphate, 1mM beta-glycerophosphate, 1mM Na3VO4And 1 mu g/mL leupeptin. CST suggested the addition of 1mM protease inhibitor (PMSF) immediately prior to use.
The primers, DNA and RNA sequences used in the kit are all synthesized in Shanghai.
A method for realizing portable rapid hybridization and detection of breast cancer related genes based on CRISPR/dcas9 magnetic nanotechnology comprises the following steps:
(1) designing sgRNA with high specificity: according to the characteristics of the breast cancer amplification target sequence, a possible sgRNA site (the PAM sequence is TGG) is analyzed by using an online website (http:// crispr. mit. edu /), and the optimal sgRNA base sequence is screened. And selecting specific sgRNA according to the difference between the paired Tm temperature value and the delta G energy value. The temperature value of target gene pairing Tm is 54.4 ℃, and the energy value of delta G is 0.33kcal/mole, so that the Tm value of the pairing region of the designed sgRNA and the target gene is higher than 54.4 ℃, and the energy value of delta G is lower than 0.33kcal/mole, and the sgRNA prepared in the way can be efficiently and specifically combined with the target HER-2/neu gene. The specificity of sgrnas was evaluated, and the sgRNA with the best specificity was selected (fig. 1). Designed in the range of about 80-150bp upstream and downstream of the selected sgRNA core sequence, and the designed dCas9 protein is subjected to SNAP-tag labeling on the dCas9 protein during the expression process.
HER-2/neu gene (SEQ ID No. 1): AH002823.2, with the thick portion being the PAM site and the underlined being the sgRNA targeting region:
dCas9-sgRNA gene sequence (SEQ ID No. 2): the sequence at the lineation is a target gene pairing region GGAGACTTC GTTGGAATGCAGTAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU
(2) Preparing the avidin magnetic nano-microsphere: weighing 2.8 × 10-3-2.9×10-3And (3) fully mixing mol/L ferric acetylacetonate and 15-25mL benzyl alcohol in an oxygen-free environment, transferring the mixture into a Teflon cup, and finally transferring the mixture into a high-pressure reaction kettle to perform reaction for 2-6h at 180 ℃ in a sealed manner better than 170-. And taking out the reaction solution after the reaction is finished, and centrifuging in a centrifugal machine to remove the supernatant to obtain the oil-soluble magnetic nanoparticles. Tttison X-100, hexanol and cyclohexane are mixed homogeneously in different proportion to form transparent stable micro emulsion system. Then adding proper amount of Fe3O4After ultrasonic treatment is carried out for 10-40min, 1-3mL of concentrated ammonia water and 1-3mL of ethyl orthosilicate are added, and the mixture is stirred for 1-3h at the temperature of 30-50 ℃. Standing and precipitating, and calcining for 2-4h at the temperature of 400-800 ℃ after cleaning by using ethanol to obtain the amino modified magnetic nanoparticles. Adding 1mg of the magnetic nanoparticles prepared by the method into a certain amount of mixed solution of methanol and glycerol, and performing ultrasonic treatment for 10-40 min. Adding a certain amount of AEAPS into the mixed solution, ultrasonically mixing uniformly, reacting for 1-3h at 80-100 ℃, taking out the particles, vacuum-drying for 1-2h by using methanol, and collecting the particles. Then packaging with streptavidin, preparing PBS buffer solution by using RNase enzyme water, and carrying out autoclaving to obtain sterile PBS. Streptavidin in an amount of 0.5-1.5mg was dissolved in 0.5mL PBS and dispensed into EP tubes at 50. mu.L per tube. 5-7mg of the treated and modified amino particles were added to 1mL of PBS and dispersed by sonication. The amino group-modified particles were suspended in 500. mu.L of PBS, 50. mu.L of the streptavidin solution was added, and the reaction was carried out with shaking at room temperature. After completion of the reaction, the particles were dispersed in 1mL of PBS solution and stored at 4 ℃ until use. FIG. 2 is an electron microscope characterization diagram of the prepared magnetic nano-microsphere.
(3) Preparing a CRISPR @ magnetic nano microsphere probe and detecting breast cancer related genes:
as shown in FIG. 3, the prepared avidin magnetic nano-microspheres are used for constructing a CRISPR @ magnetic nano-technology to realize the detection of target genes. 1-5 mu L of dCas9 protein with the concentration of 1-5 mu mol/L is taken, 1-5 mu L of SNAP-Biotin with the concentration of 1-5 mu mol/L and 10-50 mu L of avidin magnetic nano microspheres with the concentration of 1-2mg/mL are added, and the reaction is carried out for 20-50min at the temperature of 25-37 ℃. The CRISPR/dCas 9-SNAP-Biotin-magnetic nano-microsphere (CRISPR @ magnetic nano-microsphere detection probe) is prepared because the CRISPR/dCas9 protein has a SNAP label. Adding 0.5-5 mu L of guide RNA (sgRNA) with the concentration of 0.5-4 mu mol/L, complementarily pairing the sgRNA with HER-2/neu gene breast cancer mutant fragments, reacting for 20-50min at 25-37 ℃, thus quickly, sensitively and specifically binding the target gene to the surface of the CRISPR @ magnetic nanosphere detection probe, collecting the surface fluorescence signal of the magnetic nanosphere through steps of magnetic separation and washing and the like, wherein the fluorescence intensity is positively correlated with the content of the target gene, and as shown in figure 4, the CRISPR @ magnetic nanosphere is combined with HER-2 with different contents to express fluorescence display in different degrees. Thereby realizing the early screening detection and prognosis monitoring of the HER-2/neu gene of the breast cancer.
Example 1
Weighing 2.83X 10-3And (3) fully mixing mol/L ferric acetylacetonate and 20mL benzyl alcohol in an oxygen-free environment, transferring the mixture into a Teflon cup, finally transferring the Teflon cup into a high-pressure reaction kettle, and sealing the kettle to react for 2 hours at the temperature of 170 ℃. And taking out the reaction solution after the reaction is finished, and centrifuging in a centrifugal machine to remove the supernatant to obtain the oil-soluble magnetic nanoparticles. TttonX-100, hexanol and cyclohexane are uniformly mixed according to the proportion of 1:2:4 to form a transparent and stable microemulsion system. Then adding proper amount of Fe3O4After the ultrasonic treatment for 20min, 2mL of concentrated ammonia water and 2mL of ethyl orthosilicate are added, and the mixture is stirred for 2h at 30 ℃. Standing for precipitation, washing with ethanol, and calcining at 500 deg.C for 2h to obtain amino-modified magnetic nanoparticles. Adding 1mg of the magnetic nanoparticles prepared by the method into a certain amount of mixed solution of methanol and glycerol, and carrying out ultrasonic treatment for 30 min. Adding a certain amount of AEAPS into the mixed solution, mixing uniformly by ultrasound, reacting for 2h at 90 deg.C, taking out the particles, vacuum drying with methanol for 1h,the particles are collected. Then packaging with streptavidin, preparing PBS buffer solution by using RNase enzyme water, and carrying out autoclaving to obtain sterile PBS. Streptavidin (1 mg) was dissolved in 0.5mL PBS and dispensed into EP tubes at 50. mu.L per tube. 5mg of the treated and modified amino particles were added to 1mL of PBS and dispersed by sonication. The amino group-modified particles were suspended in 500. mu.L of PBS, 50. mu.L of the streptavidin solution was added, and the reaction was carried out with shaking at room temperature. After completion of the reaction, the particles were dispersed in 1mL of PBS solution and stored at 4 ℃ until use.
Example 2
Weighing 2.81X 10-3And (3) fully mixing mol/L ferric acetylacetonate and 20mL benzyl alcohol in an oxygen-free environment, transferring the mixture into a Teflon cup, finally transferring the Teflon cup into a high-pressure reaction kettle, and sealing the kettle to react for 2 hours at the temperature of 170 ℃. And taking out the reaction solution after the reaction is finished, and centrifuging in a centrifugal machine to remove the supernatant to obtain the oil-soluble magnetic nanoparticles. TttonX-100, hexanol and cyclohexane are uniformly mixed according to the proportion of 1:2:5 to form a transparent and stable microemulsion system. Then adding proper amount of Fe3O4After the ultrasonic treatment for 20min, 1mL of concentrated ammonia water and 2mL of ethyl orthosilicate are added, and the mixture is stirred for 2h at 30 ℃. Standing for precipitation, washing with ethanol, and calcining at 500 deg.C for 2h to obtain amino-modified magnetic nanoparticles. Adding 1mg of the magnetic nanoparticles prepared by the method into a certain amount of mixed solution of methanol and glycerol, and carrying out ultrasonic treatment for 30 min. Adding a certain amount of AEAPS into the mixed solution, ultrasonically mixing uniformly, reacting for 2h at 100 ℃, taking out the particles, vacuum-drying for 1h by using methanol, and collecting the particles. Then packaging with streptavidin, preparing PBS buffer solution by using RNase enzyme water, and carrying out autoclaving to obtain sterile PBS. Streptavidin (1.5 mg) was dissolved in 0.5mL of PBS and dispensed into EP tubes at 50. mu.L per tube. 5mg of the treated and modified amino particles were added to 1mL of PBS and dispersed by sonication. The amino group-modified particles were suspended in 500. mu.L of PBS, 50. mu.L of the streptavidin solution was added, and the reaction was carried out with shaking at room temperature. After completion of the reaction, the particles were dispersed in 1mL of PBS solution and stored at 4 ℃ until use.
Example 3
Six experiments, two blank groups, four experimental groups were designed. 1 mu L of dCas9 protein with the concentration of 1 mu mol/L is taken, 1 mu L of SNAP-Biotin with the concentration of 1 mu mol/L and 10 mu L of avidinated magnetic nano microspheres with the concentration of 1mg/mL are added, the mixture reacts for 25min at 28 ℃, CRISPR/dCas 9-SNAP-Biotin-magnetic nano microspheres (CRISPR @ magnetic nano microspheres) are prepared, and 1 mu L of different three groups of guide RNA (sgRNA) with the concentration of 1 mu mol/L are added after grouping. Taking 6 PCR reaction tubes, respectively subpackaging the reaction liquid into the PCR tubes, adding no gene to be detected into No.1 and No.2 centrifuge tubes, sequentially and incrementally adding target gene fragments corresponding to different concentrations into No. 3, No. 4, No. 5 and No. 6 centrifuge tubes, reacting for 25min at 28 ℃, quickly, sensitively and specifically binding the target gene fragment complemented by sgRNA to the surface of the CRISPR @ magnetic nanospheres, collecting fluorescence signals on the surfaces of the magnetic nanospheres through steps of magnetic separation and washing and the like, wherein the fluorescence intensity is positively correlated with the content of the target gene. By designing the sgRNA, early screening detection and prognosis monitoring of breast cancer HER-2/neu genes are realized.
Example 4
Six experiments, two blank groups, four experimental groups were designed. 1 mu L of dCas9 protein with the concentration of 2 mu mol/L is taken, 1 mu L of SNAP-Biotin with the concentration of 1 mu mol/L and 20 mu L of avidinated magnetic nano-microspheres with the concentration of 1mg/mL are added, the mixture reacts for 30min at 37 ℃, CRISPR/dCas 9-SNAP-Biotin-magnetic nano-microspheres (CRISPR @ magnetic nano-microspheres) are prepared, and 1 mu L of different three groups of guide RNA (sgRNA) with the concentration of 2 mu mol/L are added after grouping. Taking 6 PCR reaction tubes, respectively subpackaging the reaction liquid into the PCR tubes, adding no gene to be detected into No.1 and No.2 centrifuge tubes, sequentially and incrementally adding target gene fragments corresponding to different concentrations into No. 3, No. 4, No. 5 and No. 6 centrifuge tubes, reacting for 30min at 37 ℃, so that the target gene corresponding to sgRNA is rapidly, sensitively and specifically bound to the surface of the CRISPR @ magnetic nanospheres, collecting fluorescence signals on the surfaces of the magnetic nanospheres through steps of magnetic separation washing and the like, wherein the fluorescence intensity is positively correlated with the content of the target gene. By designing the sgRNA, early screening detection and prognosis monitoring of breast cancer HER-2/neu genes are realized.
Example 5
Six experiments, two blank groups, four experimental groups were designed. 1 mu L of dCas9 protein with the concentration of 3 mu mol/L is taken, 5 mu L of SNAP-Biotin with the concentration of 3 mu mol/L and 20 mu L of avidinated magnetic nano-microspheres with the concentration of 1mg/mL are added, the mixture reacts for 40min at 37 ℃, CRISPR/dCas 9-SNAP-Biotin-magnetic nano-microspheres (CRISPR @ magnetic nano-microspheres) are prepared, and 1 mu L of different three groups of guide RNA (sgRNA) with the concentration of 1 mu mol/L are added after grouping. Taking 6 PCR reaction tubes, respectively subpackaging the reaction liquid into the PCR tubes, adding no gene to be detected into No.1 and No.2 centrifuge tubes, sequentially and incrementally adding target gene fragments corresponding to different concentrations into No. 3, No. 4, No. 5 and No. 6 centrifuge tubes, reacting for 25min at 28 ℃, so that the target gene corresponding to sgRNA is rapidly, sensitively and specifically bound to the surface of the CRISPR @ magnetic nanospheres, collecting the fluorescence signals on the surfaces of the magnetic nanospheres through steps of magnetic separation washing and the like, wherein the fluorescence intensity is positively correlated with the content of the target gene. By designing the sgRNA, early screening detection and prognosis monitoring of breast cancer HER-2/neu genes are realized.
Example 6
Six experiments, two blank groups, four experimental groups were designed. 3 mu L of dCas9 protein with the concentration of 1 mu mol/L is taken, 1 mu L of SNAP-Biotin with the concentration of 1 mu mol/L and 10 mu L of avidin magnetic nano-microspheres with the concentration of 2mg/mL are added, the mixture reacts for 30min at 37 ℃, CRISPR/dCas 9-SNAP-Biotin-magnetic nano-microspheres (CRISPR @ magnetic nano-microspheres) are prepared, and after grouping, 3 mu L of three different sets of guide RNA (sgRNA) with the concentration of 1 mu mol/L are added. Taking 6 PCR reaction tubes, respectively subpackaging the reaction liquid into the PCR tubes, adding no gene to be detected into No.1 and No.2 centrifuge tubes, sequentially and incrementally adding target gene fragments corresponding to different concentrations into No. 3, No. 4, No. 5 and No. 6 centrifuge tubes, reacting for 30min at 37 ℃, so that the target gene corresponding to sgRNA is rapidly, sensitively and specifically bound to the surface of the CRISPR @ magnetic nanospheres, collecting fluorescence signals on the surfaces of the magnetic nanospheres through steps of magnetic separation washing and the like, wherein the fluorescence intensity is positively correlated with the content of the target gene. By designing the sgRNA, early screening detection and prognosis monitoring of breast cancer HER-2/neu genes are realized.
Sequence listing
<110> Xuzhou university of medicine
<120> gene detection method based on CRISPR/dcas9 and magnetic nano material and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 757
<212> DNA
<213> HER-2/neu Gene sequence (HER-2/neu)
<400> 1
cccgggggtc ctggaagcca caaggtaaac acaacacatc cccctccttg actatgcaat 60
tttactagag gatgtggtgg gaaaaccatt atttgatatt aaaacaaata ggcttgggat 120
ggagtaggat gcaagctccc caggaaagtt taagataaaa cctgagactt aaaagggtgt 180
taagagtggc agcctaggga atttatcccg gactccgggg gagggggcag agtcaccagc 240
ctctgcattt agggattctc cgaggaaaag tgtgagaacg gctgcaggca acccaggcgt 300
cccggcgcta ggagggacga cccaggcctg cgcgaagaga gggagaaagt gaagctggga 360
gttgccgact cccagacttc gttggaatgc agttggaggg ggcgagctgg gagcgcgctt 420
gctcccaatc acaggagaag gaggaggtgg aggaggaggg ctgcttgagg aagtataaga 480
atgaagttgt gaagctgaga ttcccctcca ttgggaccgg agaaaccagg ggagcccccc 540
gggcagccgc gcgccccttc ccacggggcc ctttactgcg ccgcgcgccc ggcccccacc 600
cctcgcagca ccccgcgccc cgcgccctcc cagccgggtc cagccggagc catggggccg 660
gagccgcagt gagcaccatg gagctggcgg ccttgtgccg ctgggggctc ctcctcgccc 720
tcttgccccc cggagccgcg agcacccaag gtgggtc 757
<210> 2
<211> 96
<212> DNA/RNA
<213> HER-2/neu Gene sequence (dCas9-sgRNA)
<400> 2
ggagacttcg ttggaatgca gtagagcuag aaauagcaag uuaaaauaag gcuaguccgu 60
uaucaacuug aaaaaguggc accgagucgg ugcuuu 96
Claims (6)
1. The gene detection method based on CRISPR/dcas9 and the magnetic nano material is characterized by comprising the following steps:
(1) obtaining a target oncogene sequence to be detected, selecting a specific sgRNA according to the difference between a paired Tm temperature value and a delta G energy value, evaluating the specificity of the sgRNA, and selecting the sgRNA with the best specificity;
(2) preparing streptavidin magnetic nano-microspheres, reacting the streptavidin magnetic nano-microspheres with SNAP-Biotin, and combining with SNAP-tag on CRISPR/dCas9 protein to form CRISPR @ magnetic nano-microspheres;
(3) and (2) incubating the prepared CRISPR @ magnetic nanospheres and the sgRNAs selected in the step (1) together, adding the incubated particles into a body fluid sample to be detected, and adding a nucleic acid dye, wherein when a target oncogene exists in the body fluid sample to be detected, the nucleic acid dye generates a fluorescence detection signal on the surface of the magnetic nanospheres, so that the target oncogene is detected.
2. The method of claim 1, wherein the preparation method of the streptavidin magnetic nanospheres comprises:
preparing superparamagnetic oil-soluble magnetic nanoparticles by adopting a high-temperature oil phase reduction method, and taking the prepared magnetic nanoparticles as magnetic cores; preparation of SiO by Water-in-oil formation of a reverse microemulsion2Magnetic nano-microsphere with core-shell structure as shell; and (3) pretreating the prepared magnetic nano-microspheres with an aminoethyl aminopropyl polydimethylsiloxane buffer solution, and then wrapping with streptavidin to form the streptavidin magnetic nano-microspheres.
3. The method of claim 1, wherein the target oncogene is HER-2/neu.
4. The method of claim 3, wherein step (1) comprises:
acquiring a sequence of HER-2/neu genes, selecting specific sgRNAs according to the difference between a pairing Tm temperature value and a delta G energy value, wherein the target gene pairing Tm temperature value is 54.4 ℃, the delta G energy value is 0.33kcal/mole, selecting sgRNAs of which the pairing region Tm value with the target gene is higher than 54.4 ℃ and the delta G energy value is lower than 0.33kcal/mole, and evaluating the specificity of the selected sgRNAs to select the sgRNAs with the best specificity.
5. Use of a method according to any one of claims 1 to 4 in the manufacture of a breast cancer detection kit or breast cancer detection apparatus.
6. A detection kit comprising the CRISPR @ magnetic nanospheres prepared in claim 1 and a selected sgRNA.
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Citations (4)
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|---|---|---|---|---|
| CN108103151A (en) * | 2017-12-08 | 2018-06-01 | 东南大学 | A kind of method and its application based on the protein-bonded detection of nucleic acids of sequence-specific nucleic acid and parting |
| CN108351350A (en) * | 2015-08-25 | 2018-07-31 | 杜克大学 | The composition and method of type endonuclease improvement genome project specificity are instructed using RNA |
| WO2019118806A1 (en) * | 2017-12-14 | 2019-06-20 | Solid Biosciences Inc. | Non-viral production and delivery of genes |
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|---|---|---|---|---|
| CN108351350A (en) * | 2015-08-25 | 2018-07-31 | 杜克大学 | The composition and method of type endonuclease improvement genome project specificity are instructed using RNA |
| CN108103151A (en) * | 2017-12-08 | 2018-06-01 | 东南大学 | A kind of method and its application based on the protein-bonded detection of nucleic acids of sequence-specific nucleic acid and parting |
| WO2019118806A1 (en) * | 2017-12-14 | 2019-06-20 | Solid Biosciences Inc. | Non-viral production and delivery of genes |
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Inventor after: Chen Minghui Inventor after: Wang Yuxin Inventor after: Tang Wuyue Inventor after: Lu Xiaotong Inventor after: Zhou Hao Inventor before: Wang Yuxin Inventor before: Chen Minghui Inventor before: Tang Wuyue Inventor before: Lu Xiaotong Inventor before: Zhou Hao |