CN111394440A - Primer group for detecting TPMT gene polymorphism and application method thereof - Google Patents
Primer group for detecting TPMT gene polymorphism and application method thereof Download PDFInfo
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Abstract
The invention provides a primer group for detecting TPMT gene polymorphism and an application method thereof, wherein the primer group comprises at least one primer pair in the following three primer pairs: the nucleotide sequence of the upstream primer for detecting the rs1800462 site of the TPMT gene is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2; the nucleotide sequence of the upstream primer for detecting the rs1800460 site of the TPMT gene is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4; the nucleotide sequence of the upstream primer for detecting the rs1142345 site of the TPMT gene is shown as SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 6. The scheme can improve the detection flux of TPMT gene polymorphism.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a primer group for detecting TPMT gene polymorphism and an application method thereof.
Background
Thioguanine and 6-mercaptopurine are widely used for induction and maintenance treatment of the stable phase of Inflammatory Bowel Disease (IBD) and are one of the most widely used drugs for treating acute lymphoblastic leukemia (A LL) in children, and precursor azathioprine plays an important role in treatment of autoimmune diseases and rejection after organ transplantation.
At present, each PCR reaction of the conventional PCR sequencing method usually aims at one polymorphic site of the TPMT gene to carry out gene mutation detection, so the detection flux of the TPMT gene polymorphism is low.
Disclosure of Invention
The embodiment of the invention provides a primer group for detecting TPMT gene polymorphism and an application method thereof, which can improve the detection flux of TPMT gene polymorphism.
In order to achieve the purpose, the invention is realized by the following technical scheme:
at present, since the rs1800462 site, rs1800460 site and rs1142345 site of the TPMT gene are major SNPs or haplotypes causing the decrease of TPMT activity, the upstream and downstream primers for specifically amplifying the hot spot mutation site region of the gene can be designed for the above-mentioned SNP sites of the TPMT gene.
The invention provides a primer group for detecting TPMT gene polymorphism, which comprises the following components: at least one of the following three primer pairs:
the nucleotide sequence of the upstream primer for detecting the rs1800462 site of the TPMT gene is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the upstream primer for detecting the rs1800460 site of the TPMT gene is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4;
the nucleotide sequence of the upstream primer for detecting the rs1142345 site of the TPMT gene is shown as SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 6.
The accuracy of TPMT gene polymorphism detection can be improved through the primer group consisting of the primer pair, and the TPMT gene polymorphism detection flux can be improved to the greatest extent.
Specifically, when the primer set is used for multiplex PCR amplification reaction, the rs1800462 site, the rs1800460 site and the rs1142345 site of the TPMT gene can be amplified in one amplification system. In general, when the rs1800462 locus of the TPMT gene is amplified by using an upstream primer SEQ ID NO.1 and a downstream primer SEQ ID NO.2, the length of a fragment of a corresponding amplification product is 587 bp; when rs1800460 of TPMT gene is amplified by using an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO.4, the length of the segment of the corresponding amplification product is 389 bp; when rs1142345 of TPMT gene is amplified by using an upstream primer SEQ ID NO.5 and a downstream primer SEQ ID NO.6, the length of the fragment of the corresponding amplification product is 1374 bp.
That is, the present embodiment can detect a nucleic acid fragment amplified by any one of the three primer pairs at a time, or can detect an accounting fragment amplified by a plurality of primer pairs at a time.
Thus, after multiplex PCR amplification is performed by using the primer sets, DNA fragments with different lengths can be generated, so that gel cutting recovery is performed on the basis of the DNA fragments with different lengths, and the nucleotide sequence is determined.
Specifically, at least one primer pair in the primer set can also be applied to preparation of a reagent or a kit for detecting the rs1800462 site, the rs1800460 site and the rs1142345 site of the TPMT gene.
Based on the above, the present invention provides an application method of a primer set for detecting polymorphism of TPMT gene, comprising:
designing the primer set of claim 1;
extracting genome DNA from a sample to be detected as an amplification template;
preparing a multiple Polymerase Chain Reaction (PCR) reaction system containing the primer group and the amplification template;
performing multiple PCR amplification reaction on the multiple PCR reaction system to obtain a PCR product;
and determining the gene mutation condition of the hot spot mutation site of the TPMT gene of the sample to be detected according to the PCR product.
In an embodiment of the present invention, the determining, according to the PCR product, a gene mutation condition of a hot spot mutation site of the TPMT gene of the sample to be tested includes:
detecting the PCR product through electrophoresis to obtain the amplified fragment size of the PCR product;
and when the amplified fragment of the PCR product is correct in size, carrying out nucleotide sequence determination on the PCR product to obtain the gene mutation condition of the hot spot mutation site of the TPMT gene of the sample to be detected.
Specifically, since the primer sets are used for performing multiplex PCR amplification, DNA fragments with different lengths can be generated, and therefore, based on the size of the amplified fragment of the PCR product, i.e., the fragment length of the PCR product, it is determined whether the amplified PCR product is a desired DNA fragment, and when the fragment length of the amplified product at the rs1800462 site is 587bp, the fragment length of the amplified product at the rs1800460 site is 389bp, and the fragment length of the amplified product at the rs1142345 site is 1374bp, the length of the amplified PCR product can be considered to be correct.
It should be noted that, since each electrophoresis detection result has a certain error, when the difference between the fragment length of the amplified product of the SNP site and the corresponding fragment length is not greater than a threshold (e.g., 20bp, 50bp), the PCR product can be sequenced.
For example, the set threshold is not greater than 40bp, and when the length of the fragment of the amplification product at the site is 564bp as a result of the actual electrophoresis and the length of the fragment of the amplification product at the site of the TPMT gene rs1800462 is 587bp, it can be considered that the length of the fragment of the amplification product at the site of the TPMT gene rs1800462 is correct, and therefore, the amplification product at the site of the TPMT gene rs1800462 is sequenced.
Specifically, agarose gel electrophoresis or polyacrylamide gel electrophoresis can be used to resolve DNA fragments of different lengths.
The application method is a method for non-diagnostic purposes.
In one embodiment of the present invention, the multiplex PCR reaction system further comprises: DNA polymerase, PCR buffer solution corresponding to the DNA polymerase, a mixture of 4 kinds of deoxyribonucleoside triphosphate dNTPs and double distilled water;
specifically, the dosage of the DNA polymerase is 0.5-5U, so that waste caused by excessive DNA polymerase is avoided and PCR reaction is inhibited while deoxynucleotides can be added to an amplification template.
Specifically, the final concentration of each dNTP is 200-500 mu M, the final concentration of each primer in the primer group is 20-300nM, and the dosage of the amplification template is 10 ng-2 mu g.
Specifically, the molar ratio of each primer pair in the primer set is: TPMT 2: TPMT 3B: TPMT3C ═ (0.1-10): (0.1-10): (0.1-10), wherein TPMT2 is a primer pair shown in SEQ ID NO.1 and SEQ ID NO.2, TPMT3B is a primer pair shown in SEQ ID NO.3 and SEQ ID NO.4, and TPMT3C is a primer pair shown in SEQ ID NO.5 and SEQ ID NO. 6.
When the primer pairs in the primer set are mixed at the above molar ratio, the amplification efficiencies (band brightnesses at each position in the electrophoretogram) of the primer pairs are substantially the same.
A molar ratio of 0.1 to 10 for TPMT2 means any value in the range of 0.1 to 10, such as 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10.
A molar ratio of 0.1 to 10 for TPMT3B means any value in the range of 0.1 to 10, such as 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10.
A molar ratio of 0.1 to 10 for TPMT3C means any value in the range of 0.1 to 10, such as 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10.
For the amount of DNA polymerase, 0.5-5U refers to any amount in the range of 0.5U to 5U, such as 0.5U, 1U, 1.5U, 2U, 2.5U, 3U, 3.5U, 4U, 4.5U, and 5U.
For each dNTP final concentration, 200 ~ 500 u M refers to 200 ~ M to 500M in the range of any concentration, for example, 200 u M, 250 u M, 300 u M, 350 u M, 400 u M, 450 u M and 500 u M.
For the final concentration of each primer, 20-300nM refers to any concentration in the range of 20nM to 300nM, e.g., 20nM, 50nM, 100nM, 150nM, 200nM, 250nM, and 300 nM.
For the amount of the template used for amplification, 10 ng-2. mu.g means any amount within the range of 10 ng-2. mu.g, for example, 10ng, 50ng, 100ng, 200ng, 300ng, 400ng, 500ng, 600ng, 700ng, 800ng, 900ng, 1. mu.g, 1.1. mu.g, 1.2. mu.g, 1.3. mu.g, 1.4. mu.g, 1.5. mu.g, 1.6. mu.g, 1.7. mu.g, 1.8. mu.g, 1.9. mu.g and 2. mu.g.
The PCR buffer solution is a concentrated buffer solution corresponding to the selected DNA polymerase, wherein the concentration degree of the PCR buffer solution can be 2 ×, 3 ×, 4 ×, 5 ×, 6 ×, 7 ×, 8 ×, 9 × or 10 ×.
In one embodiment of the present invention, the reaction conditions of the PCR reaction system are as shown in table 1:
TABLE 1
Table 1 also shows that the PCR product is preserved at 2-8 ℃.
For the temperature of the pre-denaturation under the PCR reaction conditions, 94-98 ℃ refers to any temperature in the range of 94 ℃ to 98 ℃, such as 94 ℃, 95 ℃, 96 ℃, 97 ℃ and 98 ℃.
For the time of pre-denaturation under the PCR reaction condition, 2-10 min refers to any time within 2min to 10min, such as 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min and 10 min.
For the temperature of the denaturation reaction under the PCR reaction conditions, 94-98 ℃ refers to any temperature in the range of 94 ℃ to 98 ℃, such as 94 ℃, 95 ℃, 96 ℃, 97 ℃ and 98 ℃.
For the time of denaturation under PCR reaction conditions, 10 to 90s means any time within the range of 10s to 90s, for example, 10s, 14s, 17s, 20s, 25s, 30s, 35s, 40s, 45s, 50s, 55s, 60s, 65s, 70s, 75s, 80s, 85s, and 90 s.
For the annealing temperature in the PCR reaction conditions, 55-68 ℃ means any temperature within the range of 55 ℃ to 68 ℃, for example, 55 ℃, 56 ℃, 58 ℃, 60 ℃, 62 ℃, 64 ℃, 66 ℃ and 68 ℃.
The time of the annealing reaction under the PCR reaction conditions is 10 to 90s, and is, for example, 10s, 14s, 17s, 20s, 25s, 30s, 35s, 40s, 45s, 50s, 55s, 60s, 65s, 70s, 75s, 80s, 85s, and 90 s.
For the temperature of the extension reaction under the PCR reaction conditions, 68-72 ℃ means any temperature in the range of 68 ℃ to 72 ℃, for example, 68 ℃, 69 ℃, 70 ℃, 71 ℃ and 72 ℃.
For the time of the extension reaction under the PCR reaction condition, 0.5-6 min refers to any temperature within the range of 0.5min to 6min, for example, 0.5min, 1min, 1.5min, 2min, 2.5min, 3min, 3.5min, 4min, 4.5min, 5min, 5.5min, and 6 min.
For the temperature of the final extension reaction under the PCR reaction conditions, 68-72 ℃ refers to any temperature within the range of 68 ℃ to 72 ℃, such as 68 ℃, 69 ℃, 70 ℃, 71 ℃ and 72 ℃.
For the time of the final extension reaction under the PCR reaction condition, 5-20 min refers to any time within 5min to 20min, such as 5min, 5.5min, 6min, 6.5min, 7min, 7.5min, 8min, 8.5min, 9min, 9.5min, 10min, 12min, 14min, 16min, 18min and 20 min.
Specifically, the method for extracting genomic DNA from a sample to be tested comprises the following steps: extracting by hand or kit, and extracting the extracted DNA to obtain the genome DNA.
Specifically, the sample to be tested is a sample of blood, cells, tissues or buccal swabs containing human genomic DNA.
Specifically, any primer pair shown in SEQ ID No.1 to 6 can be applied to preparation of a reagent or a kit for detecting a hot spot mutation site of a TPMT gene.
Specifically, the multiplex PCR reaction system is 10 to 100 μ l.
Compared with the prior art, the invention at least has the following beneficial effects:
(1) the interpretation of the amplification result is visual and accurate: the primers provided by the invention can ensure that the sizes of the amplification products of the primer pairs are fully distinguished, and the amplified fragments do not interact with each other to generate non-specific products or dimers, thereby ensuring the accuracy of the result.
(2) And (3) improving the detection flux: each reaction of the common PCR only aims at one primer pair to amplify to generate one nucleic acid fragment, but the multiplex PCR of the invention can simultaneously amplify at least two nucleic acid fragments, and hot spot mutation sites corresponding to at least two nucleic acid fragments of the TPMT gene can be detected through a single-tube one-time reaction.
(3) The cost is reduced: the invention can reduce the PCR reaction system from 3 systems/procedures to one system/procedure, thereby reducing the use amount of reagents and consumables such as DNA polymerase, dNTP and the like and greatly reducing the detection cost.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 shows the result of agarose gel electrophoresis detection according to an embodiment of the present invention;
FIG. 2 is a nucleotide base sequence at the polymorphic site rs1800462(G > C) and upstream and downstream thereof of the TPMT gene provided by an embodiment of the present invention;
FIG. 3 is a nucleotide base sequence at the polymorphic site rs1800460(G > A) and upstream and downstream thereof of the TPMT gene provided by an embodiment of the invention;
FIG. 4 shows nucleotide base sequences at and upstream and downstream of the polymorphic site rs1142345(A > G) of the TPMT gene provided by an embodiment of the present invention.
Detailed Description
Specifically, the reagents used in the implementation of the invention are all commercial products, and the databases used in the implementation of the invention are all public online databases. The following examples are illustrative only and are not to be construed as limiting the invention.
Example 1
Design and Synthesis of primer set
Step 1.1: based on the rs1800462 site, the rs1800460 site and the rs1142345 site of the TPMT gene, upstream and downstream primers of a specific amplification gene mutation site region are designed.
For designing the primers, Primer Quest and Primer Premier 5.0 are adopted to design the primers and carry out dimer and stem-loop mismatch analysis, the primers are designed at two ends containing mutation sites, and the annealing temperatures of 3 Primer pairs are basically kept consistent.
The primer group provided by the embodiment covers the hot spot mutation site regions of the rs1800462 site, the rs1800460 site and the rs1142345 site of the TPMT gene. Because the small nucleotide sequence change can cause the primer amplification efficiency to be obviously reduced and the specificity to be poor, the invention designs specific primers aiming at different mutations respectively, simultaneously designs a multiple PCR primer group, and synthesizes the length of a product fragment and the conditions of a gene/locus after multiple times of optimization, thereby obtaining a primer group with the optimal amplification effect. Specific primer sets are shown in table 2 below.
TABLE 2
Step 1.2: and (3) synthesizing the primer group designed in the step 1.1.
Example 2
Extraction of genomic DNA from a sample to be tested
Step 2.1: mouth shed cells or fresh peripheral blood samples were collected with mouth swabs.
And 2.2, extracting the genomic DNA from the sample by adopting a Tiangen buccal swab genomic DNA extraction kit (DP322) or a blood/cell/tissue genomic DNA extraction kit (DP304), measuring the concentration and purity of the DNA by adopting NP80-touch (IMP L EN in Germany), and storing the genomic DNA.
Example 3
Preparation of PCR reaction System
Step 3.1: and (3) taking the genome DNA obtained in the step 2.2 as an amplification template, and adopting the primer group synthesized in the step 1.2 to prepare a multiple PCR reaction system.
According to the sites listed in Table 2, 3 polymorphic sites of the TPMT gene were pooled into 1 PCR tube for multiplex PCR amplification.
Specifically, the specific composition of the reaction system is shown in table 3 below. The concentration ratio of the primer combination is as follows: TPMT 2: TPMT 3B: TPMT3C ═ 1: 1: 1.5, wherein TPMT2 is a primer pair shown in SEQ ID NO.1 and SEQ ID NO.2, TPMT3B is a primer pair shown in SEQ ID NO.3 and SEQ ID NO.4, and TPMT3C is a primer pair shown in SEQ ID NO.5 and SEQ ID NO. 6.
TABLE 3
| Reagent composition | Volume of |
| 2 × PCR buffer | 25μl |
| 2mM dNTP | 10μl |
| Primer set (10. mu.M) | 2μl |
| DNA polymerase | 2μl |
| Amplification template | 2μl |
| Double distilled water | 9μl |
The amplification reagents include PCR Buffer, dNTP, Mg2+, DNA polymerase, etc., and KOD FX enzyme system (Cat. No.: KFX-101) from Toyo Boseki (TOYOBO) is used in this example; the dosage of the DNA is 30-100 ng.
It is understood that the proportional scaling up/down of the reaction system is within the scope of the embodiments of the present invention; the amplification can also be achieved by replacing other DNA polymerase systems and adjusting the appropriate proportion.
Step 3.2: the procedure of the PCR instrument was set according to the multiplex PCR reaction conditions shown in Table 4 below, and a multiplex PCR amplification reaction was performed on the multiplex PCR reaction system prepared in step 3.1 to obtain a PCR product.
TABLE 4
It should be noted that the PCR product obtained in this example was stored at 6 ℃ until use.
Example 4
Electrophoretic detection
Step 4.1: and (3) detecting the PCR product obtained in the step 3.2 by agarose gel electrophoresis to obtain the size of the PCR product fragment.
Preparing 2% agarose gel in advance, taking 3ul of PCR product amplified in step 3.2, spotting the PCR product on the gel, applying a voltage of 120V, carrying out electrophoresis for 30min, observing the size of PCR product fragments on a gel imager (see figure 1 for details), and taking clear pictures for storage.
The detection result is shown in fig. 1, where the symbols 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 shown in fig. 1 are symbols for distinguishing the electrophoresis results of the PCR products of different samples, the left-most column of fig. 1 shows a symbol strip for characterizing the length, the right-most column of fig. 1 shows the electrophoresis result of the PCR product of the control group "10", and the middle columns show the electrophoresis results of the PCR products of different samples to be detected.
According to the comparison between the position of the bright band of each PCR product in FIG. 1 and the left-side scale bar, it can be identified which exon amplification product corresponds to each product bright band. For example, 3 bright bands from top to bottom are typically PCR products corresponding to the rs1142345 site, rs1800462 site and rs1800460 site, respectively.
Referring to fig. 1, it can be seen from the electrophoresis results of the blank set that the environmental factors have no adverse effect on the electrophoresis detection results of the sample to be detected. According to the electrophoresis result of the PCR product of each sample, 3 bright bands respectively correspond to the PCR products of the rs1142345 site, the rs1800462 site and the rs1800460 site, and the number of the bright bands is consistent with the theory; 3 bright bands are clear and have obvious intervals, different bright bands have no overlapping, no smear and the like, and the bright band effect is good. Thus, it can be shown that when the PCR amplification primer set designed in step 1.1 is used for PCR amplification, only the expected target product is generated, but no other irrelevant product is generated, and the design of the primer set is reasonable.
Example 5
Sequence determination
Step 5.1: after determining that the size of the PCR product fragment obtained in step 4.1 is correct, the PCR product obtained in step 3.3 is sent to a sequencing company for sequence determination, and a sequencing result in the format of.ab 1 is obtained.
Step 5.2: analyzing the sequencing result obtained in the step 5.1 by using Chromas sequence analysis software to obtain the polymorphism condition of 3 sites in total of the TPMT gene.
Partial sequencing results are shown in FIGS. 2-4.
FIG. 2 shows the nucleotide base sequence at the polymorphic site rs1800462(G > C) and upstream and downstream thereof of the TPMT gene. Referring to the corresponding sequencing peak plot in the frame line of FIG. 2, it can be seen that the locus rs1800462 is the GG genotype, but not the CC homozygous or GC heterozygous mutant genotype, i.e., no genetic mutation occurs at the polymorphic locus of the gene.
FIG. 3 shows the nucleotide base sequence at and upstream and downstream of the polymorphic site rs1800460(G > A) of the TPMT gene. Referring to the corresponding sequencing peak plot in the frame line of FIG. 3, it can be seen that the GA mutant heterozygote genotype with G changed into A appears at the rs1800460 site, i.e., the heterozygote mutation occurs at the gene site.
FIG. 4 shows the nucleotide base sequence at and upstream and downstream of the polymorphic site rs1142345(A > G) of the TPMT gene. Referring to the corresponding sequencing peak in the boxed line of FIG. 4, it can be seen that the AG mutation heterozygote genotype with the A-to-G mutation occurs at the rs1142345 site, i.e., the heterozygote mutation occurs at the gene site.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a" does not exclude the presence of other similar elements in a process, method, article, or apparatus that comprises the element.
Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
SEQUENCE LISTING
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<120> primer set for detecting TPMT gene polymorphism and application method thereof
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Claims (8)
1. The primer group for detecting TPMT gene polymorphism is characterized by comprising at least one primer pair of the following three primer pairs:
the nucleotide sequence of the upstream primer for detecting the rs1800462 site of the TPMT gene is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the upstream primer for detecting the rs1800460 site of the TPMT gene is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4;
the nucleotide sequence of the upstream primer for detecting the rs1142345 site of the TPMT gene is shown as SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 6.
2. The primer set of claim 1, which is used for preparing a kit for detecting the rs1800462 site, the rs1800460 site and the rs1142345 site of the TPMT gene.
3. The method for using the primer set for detecting polymorphism of TPMT gene according to claim 1, comprising:
designing the primer set of claim 1;
extracting genome DNA from a sample to be detected as an amplification template;
preparing a multiple Polymerase Chain Reaction (PCR) reaction system containing the primer group and the amplification template;
performing multiple PCR amplification reaction on the multiple PCR reaction system to obtain a PCR product;
and determining the gene mutation condition of the hot spot mutation site of the TPMT gene of the sample to be detected according to the PCR product.
4. The method of claim 3,
and determining the gene mutation condition of the hot spot mutation site of the TPMT gene of the sample to be detected according to the PCR product, wherein the gene mutation condition comprises the following steps:
detecting the PCR product through electrophoresis to obtain the amplified fragment size of the PCR product;
and when the amplified fragment of the PCR product is correct in size, carrying out nucleotide sequence determination on the PCR product to obtain the gene mutation condition of the hot spot mutation site of the TPMT gene of the sample to be detected.
5. The method according to claim 3 or 4,
the multiplex PCR reaction system further comprises: DNA polymerase, PCR buffer solution corresponding to the DNA polymerase, a mixture of 4 kinds of deoxyribonucleoside triphosphate dNTPs and double distilled water;
wherein the dosage of the DNA polymerase is 0.5-5U, the final concentration of each dNTP is 200-500 mu M, the final concentration of each primer in the primer group is 20-300nM, and the dosage of the amplification template is 10 ng-2 mu g.
6. The method of claim 5,
the DNA polymerase comprises: taq DNA polymerase, KODDNA polymerase or PfuDNA polymerase.
7. The method of claim 5,
the reaction conditions of the PCR reaction system comprise: pre-denaturation at 94-98 ℃ for 2-10 min; denaturation is carried out for 10-90 s at 94-98 ℃; annealing at 55-68 ℃ for 10-90 s; extending for 0.5-6 min at 68-72 ℃; denaturation at 94-98 ℃ for 10-90 s, annealing at 55-68 ℃ for 10-90 s and extension at 68-72 ℃ for 30-300 s, circulating for 25-40 times and extension at 68-72 ℃ for 5-20 min.
8. The method according to any one of claims 3 to 7,
the mol ratio of TPMT2, TPMT3B and TPMT3C is as follows:
TPMT 2: TPMT 3B: TPMT3C ═ (0.1-10): (0.1-10): (0.1-10), wherein TPMT2 is a primer pair shown in SEQ ID NO.1 and SEQ ID NO.2, TPMT3B is a primer pair shown in SEQ ID NO.3 and SEQ ID NO.4, and TPMT3C is a primer pair shown in SEQ ID NO.5 and SEQ ID NO. 6.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506096A (en) * | 2015-12-30 | 2016-04-20 | 广州金域检测科技股份有限公司 | Primer and method for detecting TPMT gene polymorphism |
| CN109385479A (en) * | 2018-12-30 | 2019-02-26 | 广州金域医学检验中心有限公司 | Detect the kit and method for dredging purine medicaments related gene SNP |
| CN109593854A (en) * | 2018-12-30 | 2019-04-09 | 广州金域医学检验集团股份有限公司 | Nucleic acid group and application suitable for the detection of tumour class pharmaceutical relevant gene SNP site |
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2020
- 2020-04-30 CN CN202010365808.5A patent/CN111394440A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506096A (en) * | 2015-12-30 | 2016-04-20 | 广州金域检测科技股份有限公司 | Primer and method for detecting TPMT gene polymorphism |
| CN109385479A (en) * | 2018-12-30 | 2019-02-26 | 广州金域医学检验中心有限公司 | Detect the kit and method for dredging purine medicaments related gene SNP |
| CN109593854A (en) * | 2018-12-30 | 2019-04-09 | 广州金域医学检验集团股份有限公司 | Nucleic acid group and application suitable for the detection of tumour class pharmaceutical relevant gene SNP site |
Non-Patent Citations (1)
| Title |
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| SILVIA JIMÉNEZ-MORALES ET AL.: "Analysis of Thiopurine S-Methyltransferase Deficient Alleles in Acute Lymphoblastic Leukemia Patients in Mexican Patients.", 《ARCH MED RES.》 * |
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