CN110699445A - AS3MT gene single nucleotide polymorphism site detection kit, use method and application - Google Patents
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Abstract
The invention relates to a kit for detecting single nucleotide polymorphism sites of AS3MT gene, a using method and application thereof, belonging to the technical field of medicine and health. In order to solve the problem that the occurrence of the high leucocythemia cannot be predicted in the prior art, the invention provides an AS3MT gene single nucleotide polymorphism site detection kit, which comprises a specific primer for detecting rs3740390 polymorphism site on an AS3MT gene. Through PCR amplification, purification, single base extension of rs3740390 polymorphic site and purification, sequencing and result analysis of extension products, whether the detected sample has the susceptibility to the high leucocythemia can be quickly and accurately predicted according to the genotype of the polymorphic site. The application of the method in predicting the occurrence of the high leucocythemia in the process of treating the APL by the arsenic medicament can realize the individual scientific medication of the arsenic medicament, effectively reduce the occurrence rate and the fatality rate of the high leucocythemia, and has very important clinical significance.
Description
Technical Field
The invention belongs to the technical field of medicine and health, and particularly relates to a kit for detecting single nucleotide polymorphism sites of AS3MT gene, a use method and application thereof.
Background
The high leucocythemia is critical state of hematology, and means that the number of peripheral blood leucocytes of a patient is more than 10 x 109And L. The patient is easy to have life-threatening complications such as intracranial hemorrhage, cerebral infarction, adult respiratory distress syndrome, tumor cytolysis and the like, and early death is easy to occur.
Acute Promyelocytic Leukemia (APL) is a malignant clonal disease of hematopoietic stem cells. Single dose arsenic agents have low recurrence rate and high remission rate in the treatment of initial and recurrent APL patients, and are the first-line first-choice drugs for APL treatment. However, the arsenic agent for treating APL is a double-edged sword, and when the APL achieves remarkable curative effect, part of patients can have the high leucocythemia in the clinical application process, and the early survival rate and the survival quality of the patients are seriously threatened.
The research proves that the treatment and toxic and side effects of the arsenic agent depend on the active metabolites generated by the arsenic agent, but the methylation metabolic capacity of the arsenic has large difference among patients, and the difference can directly influence the treatment effect and the occurrence of complications generated in the treatment process of the individuals. The individualized differences in the treatment of arsenic and the occurrence of complications in APL patients pose significant challenges to the rational clinical administration of APL.
With the development of clinical pharmacogenomics, the aim of predicting the curative effect and toxicity of a medicament can be fulfilled by detecting Single Nucleotide Polymorphism (SNP) of a patient-related gene, so that a medicament treatment scheme is optimized. As3MT gene is located in the 10q24.32 region of chromosome 10, and various genetic polymorphisms, including SNPs and variable-number tandem repeats, have been identified in this gene. According to epidemiological studies, the polymorphism of AS3MT gene is closely related to the metabolism of arsenic in vivo and the occurrence of diseases. Although studies on AS3MT gene polymorphism and arsenic methylation metabolism have been made with considerable success in genomics, the relationship between the As3MT gene polymorphism site and the occurrence of hyperleukocythemia in APL patients treated with arsenic agents is poorly understood.
Disclosure of Invention
In order to solve the problem that the susceptibility to the high leucocythemia cannot be predicted in the prior art, the invention provides a single nucleotide polymorphism site detection kit of an AS3MT gene, a use method and application thereof.
The technical scheme of the invention is as follows:
a kit for detecting the single nucleotide polymorphism site of the AS3MT gene comprises specific primers for detecting the rs3740390 polymorphism site on the AS3MT gene, wherein the specific primers comprise a primer for amplifying the rs3740390 polymorphism site and a primer for extending the rs3740390 polymorphism site by a single base.
The rs3740390 locus on the AS3MT gene is 102878723 th nucleotide from the 5' end in chromosome 10 of a human genome; the nucleotide at the rs3740390 site is C/T.
Further, the primer sequence for amplifying the rs3740390 polymorphic site is as follows:
an upstream primer: 5'-TCTTGCCTCCTGTACAATGG-3', Seq ID No. 1;
a downstream primer: 5'-CAGAAAAATGGGAGGCAATG-3', Seq ID No. 2.
Further, the primer sequence of the single-base extension rs3740390 polymorphic site is as follows:
5'-TTTTTTGTAAATAGAGTGAAGTGCT-3', Seq ID No. 3.
Further, the method also comprises the following reagents: DEPC water, Quick Taq HS DyeMix, PCR product purification enzyme, single-base extension reaction enzyme, corresponding buffer solution and single-base extension product purification enzyme.
The application method of the AS3MT gene single nucleotide polymorphism site detection kit provided by the invention comprises the following steps:
step one, collecting peripheral venous blood and extracting genome DNA from the peripheral venous blood;
secondly, PCR amplification of the rs3740390 polymorphic site on the AS3MT gene is carried out by using a primer for amplifying the rs3740390 polymorphic site and taking the genome DNA AS a template;
step three, purifying the PCR amplification product obtained in the step two;
step four, extending the single base of the rs3740390 polymorphic site by using a primer of the single base extended rs3740390 polymorphic site and taking the purified PCR amplification product obtained in the step three as a template;
step five, purifying the single base extension product obtained in the step four;
and step six, sequencing the purified single-base extension product obtained in the step five.
Further, the reaction system of the PCR amplification in the second step is: DEPC water 16 u l, Quick Taq HSDyemix25 u l, genomic DNA template 7 u l, upstream primer 1 u l and downstream primer 1 u l.
And further, the purification method of the PCR amplification product in the third step is to mix shrimp alkaline phosphatase and exonuclease I according to the volume ratio of 1:1 to prepare a digestive juice, add the digestive juice into the PCR amplification product, perform warm bath for 60min at 37 ℃, and inactivate for 15min at 80 ℃.
Further, the reaction system of the single base extension in the fourth step is: 2 μ l of the purified PCR amplification product template obtained in the third step, 2 μ l of the primer of the single base extension rs3740390 polymorphic site in the fourth step, 0.8 μ l of BigDye, 1.6 μ l of Sequencing Buffer and 3.6 μ l of DEPC water.
Furthermore, the purification method of the single base extension product in the fifth step is to add shrimp alkaline phosphatase into the single base extension product, perform warm bath at 37 ℃ for 60min, and inactivate at 75 ℃ for 15 min.
The AS3MT gene single nucleotide polymorphism site detection kit provided by the invention is applied to prediction of susceptibility of the leukemia, in particular to prediction of susceptibility of the leukemia in the process of treating APL by an arsenic agent.
The arsenic agent of the invention comprises arsenous acid, compound Huangdai tablets and other arsenic agents used for treating APL.
The risk of generating the high leucocythemia of the APL patient to be detected with the CC genotype at the rs3740390 locus on the AS3MT gene treated by the arsenic agent is higher than that of the APL patient to be detected with the CT genotype or the TT genotype at the rs3740390 locus.
The invention has the beneficial effects that:
the invention provides a kit for detecting single nucleotide polymorphism sites of AS3MT gene and a using method thereof, which can quickly and accurately detect the genotype of rs3740390 polymorphism sites on AS3MT gene. The method is applied to predicting the susceptibility of the arsenic agent to the high leucocythemia in the APL treatment process, and whether the arsenic agent has higher susceptibility to the high leucocythemia after being treated by the arsenic agent can be accurately predicted by detecting the genotype of the rs3740390 polymorphic site on the AS3MT gene of a patient, so that the individual scientific medication of the arsenic agent is realized, the incidence rate and the fatality rate of the high leucocythemia in the APL treatment process by the arsenic agent are effectively reduced, and the method has very important clinical significance.
Drawings
FIG. 1 is a diagram of DNA sequencing analysis of a single-base extension product,
the dotted line frame in the figure shows the rs3740390 polymorphic site on the AS3MT gene, the rs3740390 polymorphic site shown in the figure A is CC type, the rs3740390 polymorphic site shown in the figure B is CT type, and the rs3740390 polymorphic site shown in the figure C is TT type;
FIG. 2 is a graph comparing the relationship between AS3MT rs3740390 and the leukopenia.
Detailed Description
The technical solutions of the present invention are further described below with reference to the following examples, but the present invention is not limited thereto, and any modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Example 1
A kit for detecting the single nucleotide polymorphism sites of AS3MT gene comprises the following reagents:
(1) a specific primer for detecting the rs3740390 site on the AS3MT gene;
(2) DEPC water;
(3)Quick Taq HS DyeMix；
(4) purifying the enzyme by using a PCR product;
(5) single base extension reaction enzyme and corresponding buffer solution;
(6) the single base extension product purified the enzyme.
The specific primers for detecting the rs3740390 polymorphic site on the AS3MT gene comprise primers for amplifying the rs3740390 polymorphic site and primers for extending the rs3740390 polymorphic site by single base.
In this embodiment, the primer sequence for amplifying the rs3740390 polymorphic site is as follows:
an upstream primer: 5'-TCTTGCCTCCTGTACAATGG-3', Seq ID No. 1;
a downstream primer: 5'-CAGAAAAATGGGAGGCAATG-3', Seq ID No. 2.
The primer sequence of the single-base extension rs3740390 polymorphic site is as follows:
5'-TTTTTTGTAAATAGAGTGAAGTGCT-3', Seq ID No. 3.
In this embodiment, the design of the primer is performed for the AS3MT gene, and the specific process is AS follows: the sequence of the AS3MT gene in the primate mRNA sequence was looked up at NCBI (AY817668), primers were designed using Primer 5.0 software, the highest scoring primers were selected from them, the designed primers were evaluated using Oligo 6.0 software, the specificity of the primers was checked by Blast at NCBI website, and qualified Primer sequences were submitted to Thermo Fisher Scientific (Invitrogen) for preparation.
The rs3740390 locus on the AS3MT gene is 102878723 th nucleotide from the 5' end in chromosome 10 of a human genome; the nucleotide at the rs3740390 site is C/T.
Example 2
This example is a method for using the kit for detecting a single nucleotide polymorphism of AS3MT gene provided in example 1, and the steps are AS follows:
step one, collecting peripheral venous blood and extracting genome DNA from the peripheral venous blood;
secondly, PCR amplification of the rs3740390 polymorphic site on the AS3MT gene is carried out by using a primer for amplifying the rs3740390 polymorphic site and taking the genome DNA AS a template;
step three, purifying the PCR amplification product obtained in the step two;
step four, extending the single base of the rs3740390 polymorphic site by using a primer of the single base extended rs3740390 polymorphic site and taking the purified PCR amplification product obtained in the step three as a template;
step five, purifying the single base extension product obtained in the step four;
and step six, sequencing the purified single-base extension product obtained in the step five.
Example 3
This example provides a specific method for collecting peripheral venous blood and extracting genomic DNA therefrom, comprising the steps of:
1. collecting blood specimen of APL patient
The patients were the initial APL patients who met the Chinese guidelines for the diagnosis and treatment of acute promyelocytic leukemia (2018 edition). 2ml of peripheral venous blood of the patient was collected in blood collection tubes containing EDTA.
2. Extraction of genomic DNA from a patient
The genomic DNA of the patient was extracted from 200. mu.l of peripheral blood of the patient using a whole genomic DNA extraction kit (product of Biochemical Co., Ltd., Beijing Tian, Ltd.) according to the instructions. The specific operation flow is as follows:
(1) 200 mul of peripheral venous blood of a patient is taken, 20 mul of protein kinase K is added and mixed evenly.
(2) Add 200. mu.l buffer GB, mix well by inversion, incubate 10min at 56 ℃ while mixing by inversion several times, the solution should be clear.
(3) Add 200. mu.l of absolute ethanol and mix well by inversion.
(4) The solution and flocculent precipitate obtained in the previous step are added into an adsorption column CB3, centrifuged at 12000rpm for 30sec, and the waste liquid in the collecting tube is poured off.
(5) To the adsorption column CB3, 500. mu.l of buffer GD was added, centrifuged at 12000rpm for 30sec, and the waste liquid in the collection tube was discarded.
(6) 600. mu.l of the rinsing solution PW was added to the adsorption column CB3, and centrifuged at 12000rpm for 30sec to discard the waste liquid in the collection tube.
(7) And (6) repeating the step.
(8) And (4) pouring the waste liquid in the collecting pipe.
(9) Centrifuge at 12,000rpm for 2min and discard the waste. The adsorption column CB3 was left at room temperature for several minutes.
(10) Transferring the adsorption column CB3 into a 1.5ml centrifuge tube, suspending and dripping 50 elution buffer TB into the middle position of the adsorption membrane, incubating at room temperature for 5min, centrifuging at 12000rpm for 2min, and collecting the solution into the centrifuge tube to obtain the product.
Example 4
The present embodiment provides a method for performing PCR amplification and product purification on the rs3740390 polymorphic site on the AS3MT gene by using the primer for amplifying the rs3740390 polymorphic site and using the genomic DNA extracted in embodiment 3 AS a template, comprising the following steps:
(1) patient DNA concentration assessment
Mu.l of the patient DNA sample obtained in example 3 was used for DNA concentration and purity determination on a NanoDrop spectrophotometer (ND-1000, ThermoFisher Scientific):
DNA purity: DNA should have a significant absorption peak at OD260, the ratio OD260/OD280 should be 1.7-1.9,
DNA concentration: greater than 30 ng/. mu.l.
(2) PCR amplification reaction of target gene locus
A primer for amplifying the rs3740390 polymorphic site:
5’-TCTTGCCTCCTGTACAATGG-3’(Seq ID No.1),
5’-CAGAAAAATGGGAGGCAATG-3’(Seq ID No.2)；
a50. mu.l PCR reaction was prepared as shown in Table 1:
TABLE 1
| Name of reagent | Dosage (mu l) |
| DEPC water | 16 |
| Quick Taq HS DyeMix | 25 |
| DNA template | 7 |
| Primer(Seq ID No.1) | 1 |
| Primer(Seq ID No.2) | 1 |
DEPC water in Table 1 was obtained from Invitrogen, Quick Taq HS DyeMix was obtained from TOYOBO.
Setting a PCR amplification reaction program:
the first step is as follows: activating the DNA polymerase: multiplying at 95 ℃ for 3 min;
the second step is that: denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 35 s. Circulating for 35 times;
the third step: extension at 72 ℃ for 2min and holding at 4 ℃.
(3) Purification of PCR products
The digests were prepared as shown in table 2:
TABLE 2
| Reagent | Dosage (mu l) |
| Shrimp alkaline phosphatase | 1 |
| Exonuclease I | 1 |
In Table 2, Shrimp Alkaline Phosphatase (SAP) and exonuclease I (Exonuclease I) were purchased from ABI.
And (3) purification reaction:
and (3) adding 2 mul of digestive juice into the PCR amplification product obtained in the step (2), placing the PCR amplification product in a PCR instrument for warm bath for 60min at the temperature of 37 ℃, inactivating the PCR amplification product for 15min at the temperature of 80 ℃ to obtain a purified PCR amplification product, and keeping the purified PCR amplification product at the temperature of 4 ℃.
Example 5
This example provides a method for single base extension of rs3740390 polymorphic site and a method for purification of the extended product using the PCR amplified product obtained in example 4 as a template, using a primer for single base extension of the rs3740390 polymorphic site, comprising the following steps;
(1) single base extension reaction
Primer for single-base extension of rs3740390 polymorphic site:
5’-TTTTTTGTAAATAGAGTGAAGTGCT-3’(Seq ID No.3)；
single base extension reaction conditions:
a single-base extension reaction system (10. mu.l) was prepared as shown in Table 3:
TABLE 3
| Name of reagent | Dosage (mu l) |
| Purified PCR product | 2 |
| Primer(Seq ID No.3) | 2 |
| Sequencing Buffer | 1.6 |
| BigDye | 0.8 |
| DEPC Water (Invitrogen) | 3.6 |
In Table 3, Sequencing Buffer and BigDye are ABI PRISMTMBigDyeTMThe Terminator v3.1cycle Sequencing Kit.
Setting a single base extension reaction program:
the first step is as follows: pre-denaturation at 96 ℃ for 2 min;
the second step is that: 96 ℃ X10 s,55 ℃ X5 s,60 ℃ X90 s. A total of 25 cycles;
the third step: kept at 4 ℃.
(2) Purification of the extension product:
adding 1 μ l of shrimp alkaline phosphatase into 10 μ l of the extension product, performing warm bath at 37 deg.C for 60min, and inactivating at 75 deg.C for 15min to obtain purified single base extension product.
Example 6
In this example, the purified single-base extension product obtained in example 5 is sequenced and analyzed for results, and susceptibility to hyperleukocythemia is predicted, specifically as follows:
(1) sanger sequencing was performed using an ABI PRISM 3730XL sequencer;
(2) genotyping: and analyzing a sequencing result by using Chromas Pro2.4.1 software, and judging the rs3740390 polymorphic site genotype.
As shown in fig. 1, when the sequencing result is shown in fig. a, the genotype of the rs3740390 polymorphic site is the CC genotype; when the sequencing result is shown in a graph B, the genotype of the rs3740390 polymorphic site is a CT genotype; when the sequencing result is shown in a figure C, the genotype of the rs3740390 polymorphic site is TT genotype.
Example 7
The AS3MT gene single nucleotide polymorphism site detection kit provided by the invention is used for detecting the genotypes of the AS3MT rs3740390 polymorphism sites of 74 cases of initial APL patients adopting arsenious acid single-agent continuous slow-point therapy, and all tested patients successfully complete induction and relief, wherein 36 patients have hyperleukocythemia in the treatment process, and 38 patients have no hyperleukocythemia.
Analysis of the AS3MT rs3740390 polymorphic site in the above 74 patients showed that the genotype distributions of the polymorphic sites all meet Hardy-Weinburg equilibrium, and the results are shown in Table 4.
TABLE 4
Since the number of patients in homozygous mutant type is small, patients are divided into two groups of mutant type (heterozygous + homozygous mutant type) and wild type. Statistical analysis shows that the occurrence of the high leucocythemia in the APL patients treated by the arsenic agent has correlation with the polymorphism of AS3MT rs3740390 site (P <0.01), and the result is shown in figure 2. Therefore, the AS3MT rs3740390 site polymorphism can predict the susceptibility of the APL patients to the high leucocythemia in the arsenic treatment process. Wherein, the sample with CC genotype at rs3740390 site on AS3MT gene has higher risk of generating high leucocythemia than the sample with CT genotype or TT genotype at rs3740390 site. Namely, when the AS3MT rs3740390 polymorphism locus genotype of the patient is CC, the patient is predicted to have higher susceptibility to the high leucocythemia.
The AS3MT gene single nucleotide polymorphism site detection kit provided by the invention is used for predicting the susceptibility of the APL patient receiving arsenic agent treatment to the high leucocythemia, and the detection of the genotype of the rs3740390 polymorphism site on the AS3MT gene of the patient can accurately predict whether the patient has higher susceptibility of the high leucocythemia after being treated by the arsenic agent, thereby realizing the individualized scientific medication of the arsenic agent, effectively reducing the incidence and the fatality rate of the high leucocythemia in the process of treating the APL by the arsenic agent and having very important clinical significance.
SEQUENCE LISTING
<110> Harbin university of medicine
<120> AS3MT gene single nucleotide polymorphism site detection kit, using method and application
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ttttttgtaa atagagtgaa gtgct 25
Claims (10)
1. A kit for detecting the single nucleotide polymorphism site of the AS3MT gene is characterized by comprising specific primers for detecting the rs3740390 polymorphism site on the AS3MT gene, wherein the specific primers comprise a primer for amplifying the rs3740390 polymorphism site and a primer for extending the rs3740390 polymorphism site by a single base.
2. The kit for detecting the AS3MT gene single nucleotide polymorphism site according to claim 1, wherein the primer sequence for amplifying the rs3740390 polymorphism site is AS follows:
an upstream primer: 5'-TCTTGCCTCCTGTACAATGG-3', Seq ID No. 1;
a downstream primer: 5'-CAGAAAAATGGGAGGCAATG-3', Seq ID No. 2.
3. The kit for detecting the AS3MT gene single nucleotide polymorphism site according to claim 1 or 2, wherein the primer sequence of the single base extension rs3740390 polymorphism site is AS follows:
5'-TTTTTTGTAAATAGAGTGAAGTGCT-3', Seq ID No. 3.
4. The kit for detecting the single nucleotide polymorphism site of AS3MT gene according to claim 3, further comprising the following reagents: DEPC water, Quick Taq HSDyemix, a PCR product purifying enzyme, a single-base extension reaction enzyme and corresponding buffer solution and a single-base extension product purifying enzyme.
5. A method for using the kit for detecting the single nucleotide polymorphism site of AS3MT gene according to any one of claims 1-4, which comprises the following steps:
step one, collecting peripheral venous blood and extracting genome DNA from the peripheral venous blood;
secondly, PCR amplification of the rs3740390 polymorphic site on the AS3MT gene is carried out by using a primer for amplifying the rs3740390 polymorphic site and taking the genome DNA AS a template;
step three, purifying the PCR amplification product obtained in the step two;
step four, extending the single base of the rs3740390 polymorphic site by using a primer of the single base extended rs3740390 polymorphic site and taking the purified PCR amplification product obtained in the step three as a template;
step five, purifying the single base extension product obtained in the step four;
and step six, sequencing the purified single-base extension product obtained in the step five.
6. The method for using the AS3MT gene SNP site detection kit according to claim 5, wherein the reaction system of the PCR amplification in the second step is: DEPC water 16 u l, Quick Taq HSDyemix25 u l, genomic DNA template 7 u l, upstream primer 1 u l and downstream primer 1 u l.
7. The use method of the kit for detecting the single nucleotide polymorphism of the AS3MT gene according to claim 5 or 6, wherein the purification method of the PCR amplification product in the third step is to mix shrimp alkaline phosphatase and exonuclease I according to the volume ratio of 1:1 to prepare a digestive juice, add the digestive juice into the PCR amplification product, perform warm bath at 37 ℃ for 60min, and inactivate at 80 ℃ for 15 min.
8. The method for using the kit for detecting the single nucleotide polymorphism of AS3MT gene according to claim 7, wherein the reaction system for single base extension in step four is: 2 μ l of the purified PCR amplification product template obtained in the third step, 2 μ l of primers for single base extension of the rs3740390 polymorphic site, 0.8 μ l of BigDye, 1.6 μ l of Sequencing Buffer and 3.6 μ l of DEPC water.
9. The method for using the kit for detecting the single nucleotide polymorphism of AS3MT gene according to claim 8, wherein the purification method of the single base extension product in step five is to add shrimp alkaline phosphatase into the single base extension product, incubate at 37 ℃ for 60min, and inactivate at 75 ℃ for 15 min.
10. Use of the kit for detecting the single nucleotide polymorphism of AS3MT gene according to any one of claims 1-4 for predicting susceptibility to hyperleukocythemia.
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| CN113528661A (en) * | 2021-02-22 | 2021-10-22 | 北京市理化分析测试中心 | Primer for amplifying CTLA4 gene SNP site, detection kit and application |
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| CN113528661A (en) * | 2021-02-22 | 2021-10-22 | 北京市理化分析测试中心 | Primer for amplifying CTLA4 gene SNP site, detection kit and application |
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