CN111534591A - Method for synchronously detecting gene polymorphism of two SNP sites of UGT1A1 gene - Google Patents
Method for synchronously detecting gene polymorphism of two SNP sites of UGT1A1 gene Download PDFInfo
- Publication number
- CN111534591A CN111534591A CN202010369763.9A CN202010369763A CN111534591A CN 111534591 A CN111534591 A CN 111534591A CN 202010369763 A CN202010369763 A CN 202010369763A CN 111534591 A CN111534591 A CN 111534591A
- Authority
- CN
- China
- Prior art keywords
- ugt1a1
- gene
- pcr
- primer
- snp sites
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 101710205316 UDP-glucuronosyltransferase 1A1 Proteins 0.000 title claims abstract description 59
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims description 15
- 102100029152 UDP-glucuronosyltransferase 1A1 Human genes 0.000 claims abstract description 38
- 238000001514 detection method Methods 0.000 claims abstract description 27
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- 238000012408 PCR amplification Methods 0.000 claims abstract description 12
- 101000841498 Homo sapiens UDP-glucuronosyltransferase 1A1 Proteins 0.000 claims abstract description 10
- 239000002773 nucleotide Substances 0.000 claims abstract description 10
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 10
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 10
- 230000003321 amplification Effects 0.000 claims description 13
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 13
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 11
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 238000007400 DNA extraction Methods 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 102000054765 polymorphisms of proteins Human genes 0.000 claims description 5
- 238000000137 annealing Methods 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 4
- 239000012498 ultrapure water Substances 0.000 claims description 4
- 238000012257 pre-denaturation Methods 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- 239000012634 fragment Substances 0.000 abstract description 10
- 230000004907 flux Effects 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 41
- 108020004414 DNA Proteins 0.000 description 17
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 9
- 238000012163 sequencing technique Methods 0.000 description 7
- FJHBVJOVLFPMQE-QFIPXVFZSA-N 7-Ethyl-10-Hydroxy-Camptothecin Chemical compound C1=C(O)C=C2C(CC)=C(CN3C(C4=C([C@@](C(=O)OC4)(O)CC)C=C33)=O)C3=NC2=C1 FJHBVJOVLFPMQE-QFIPXVFZSA-N 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 229960004768 irinotecan Drugs 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 5
- 206010012735 Diarrhoea Diseases 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 206010064571 Gene mutation Diseases 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 210000000941 bile Anatomy 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000002207 metabolite Substances 0.000 description 3
- 208000004235 neutropenia Diseases 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 108700028369 Alleles Proteins 0.000 description 2
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- 102000004308 Carboxylic Ester Hydrolases Human genes 0.000 description 2
- 108090000863 Carboxylic Ester Hydrolases Proteins 0.000 description 2
- 206010052358 Colorectal cancer metastatic Diseases 0.000 description 2
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- SSJQVDUAKDRWTA-CAYKMONMSA-N SN38 glucuronide Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1O[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O SSJQVDUAKDRWTA-CAYKMONMSA-N 0.000 description 2
- 108010020961 UGT1A1 enzyme Proteins 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940097043 glucuronic acid Drugs 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- -1 phenolic compounds anthraquinone Chemical class 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010062427 GDP-mannose 4,6-dehydratase Proteins 0.000 description 1
- 102000002312 GDPmannose 4,6-dehydratase Human genes 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 108010092364 Glucuronosyltransferase Proteins 0.000 description 1
- 102000016354 Glucuronosyltransferase Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 108700026226 TATA Box Proteins 0.000 description 1
- 108010067922 UDP-Glucuronosyltransferase 1A9 Proteins 0.000 description 1
- 101710205340 UDP-glucuronosyltransferase 1A7 Proteins 0.000 description 1
- 102100040213 UDP-glucuronosyltransferase 1A7 Human genes 0.000 description 1
- 102100040212 UDP-glucuronosyltransferase 1A9 Human genes 0.000 description 1
- XCCTYIAWTASOJW-XVFCMESISA-N Uridine-5'-Diphosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 XCCTYIAWTASOJW-XVFCMESISA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 208000027119 bilirubin metabolic disease Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 208000036796 hyperbilirubinemia Diseases 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000989 no adverse effect Toxicity 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 206010043554 thrombocytopenia Diseases 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a primer pair and a detection method for synchronously detecting two SNP sites of UGT1A1 gene, which are beneficial to improving detection flux. The invention provides a primer pair for synchronously detecting gene polymorphism of two SNP sites of UGT1A1 gene, which comprises a pair of primers, wherein the nucleotide sequence of an upstream primer is shown by SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown by SEQ ID NO. 2. When the primer pair is used for PCR amplification reaction, UGT1A1 × 28(-53TA6 > TA7) and UGT1A1 × 6(211G > A) sites of UGT1A1 gene are simultaneously amplified, namely, fragments containing 2 SNP sites are simultaneously amplified. The invention can detect the gene polymorphism of UGT1A 1X 28(-53TA6 & gtTA 7) and UGT1A 1X 6(211G & gtA) sites of UGT1A1 gene by one reaction, can detect more than 90 samples at the same time, not only improves the detection efficiency, but also greatly saves the cost.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a primer pair and a detection method for synchronously detecting gene polymorphism of two SNP sites of UGT1A1 gene.
Background
The human UGT1A1 gene is located in 2q37 of chromosome 2, has a span of about 160kb, and forms 9 functional transcription products by different splicing modes, namely UGT1A1-UGT1A9, wherein the most common are UGT1A1, UGT1A7 and UGT1A9, and the single nucleotide polymorphisms of UGT1A1 are mainly UGT1A 1-28 (-53TA6 > TA7) and UGT1A 1-6 (211G > A). These two variants are a combination of any two alleles: UGT1a1 × 28 refers to the combination of TA sequences at position 28 of the promoter region of UGT1a1 gene, with 6 repeats of TA sequences being × 1/× 1 (homozygous wild type), 6 and 7 repeats being × 1/× 28 (heterozygous mutant), and 7 repeats being × 28/× 28 (homozygous mutant). UGT1A1 × 6 (exon 211 site No. 1) GG type is homozygous wild type, GA type is heterozygous mutant type, and AA type is homozygous mutant type. The UGT1A1 gene is expressed in human liver, bile tissue, large intestine and stomach, and is mainly present in human liver and responsible for bilirubin glucuronic acid binding. This activity can cause hyperbilirubinemia, and can also increase the water solubility of phenolic compounds anthraquinone, flavonoid, and various drugs and their metabolites in vivo, so that the low substances are dissolved and then discharged out of the body with bile or urine.
Irinotecan (CPT-11) is one of the most effective chemotherapeutic drugs for metastatic colorectal cancer (mCRC), and delayed diarrhea and neutropenia are common adverse reactions caused by irinotecan, with a clinical incidence of 46% and 30%, respectively, and severe cases of patient death.
CPT-11 is converted to 7-ethyl-10-hydroxycamptothecin (SN-38) by hydrolysis with Carboxylesterases (CEs) in vivo. Irinotecan is converted into an active metabolite SN-38 with stronger cytotoxicity in vivo after intravenous injection, is inactivated into a glucuronic acid product (10-O-glucuronic-SN-38, SN-38G) through uridine diphosphate glucuronosyl transferase families (UGTs), is excreted into intestines through bile, and can cause intestinal mucosa injury and delayed diarrhea after being converted into SN-38 again under the action of intestinal bacteria beta-glucuronidase; UGTs enzyme in intestinal tract can catalyze SN-38 to detoxify SN-38G again. The presence of the UGT1a1 x 28 allele in CPT-11 treatment resulted in a significant increase in the active metabolite SN-38, and thus a significant increase in the incidence of diarrhea or neutropenia. Meanwhile, UGT1a1 x 6 (exon 211 1) heterozygous mutant G/a and homozygous mutant a/a significantly increased the risk of grade 3-4 neutropenia, thrombocytopenia and diarrhea in patients treated with irinotecan in asian populations.
By detecting gene polymorphism of TATA box region (UGT1A1 x 28) of promoter region of human UGT1A1 gene and nucleotide 211 of exon 1 (UGT1A1 x 6), toxic and side effects of irinotecan on tumor patients are predicted from gene level, and reference is provided for clinical doctors to formulate administration dosage of irinotecan.
PCR (Polymerase Chain Reaction) has been widely used in medicine, genetics, microbiology, and even throughout life sciences. At present, PCR detection experiments can be respectively designed aiming at two polymorphic sites UGT1A1 x 28(-53TA6 > TA7) and UGT1A1 x 6(211G > A) of UGT1A1 gene, so as to detect gene mutation conditions. Since each PCR reaction is directed to only one exon/site, the detection throughput is low.
Disclosure of Invention
In order to solve the above defects, the technical object of the present invention is to provide a primer pair and a detection method for synchronously detecting two SNP sites of UGT1a1 gene, which are beneficial to improving detection flux.
In order to achieve the purpose, the invention is realized by the following technical scheme:
firstly, a primer pair for synchronously detecting gene polymorphism of two SNP sites of UGT1A1 gene is provided, which comprises a pair of primers, wherein the nucleotide sequence of an upstream primer is shown by SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown by SEQ ID NO. 2. The primer pair is specially designed for UGT1A1 x 28(-53TA6 > TA7) and UGT1A1 x 6(211G > A) site-specific amplification of UGT1A1 genes, can simultaneously, synchronously, efficiently, specifically and accurately amplify a section of specific genes of the genes at the sites, and detects the gene polymorphism of the genes by sequencing. When the primer pair is used for PCR amplification reaction, UGT1A1 × 28(-53TA6 > TA7) and UGT1A1 × 6(211G > A) sites of UGT1A1 gene are simultaneously amplified, namely, fragments containing 2 SNP sites are simultaneously amplified. The detection flux can be improved to the maximum extent.
Multiple experiments prove that the primer pair provided by the invention has better specificity and amplification accuracy, can be applied to preparing a kit for synchronously detecting the gene polymorphism of two SNP sites of UGT1A1 gene, and can quickly and accurately obtain the gene polymorphism results of UGT1A1 gene at two sites after sample experiments are collected by preparing a finished kit.
The kit mainly comprises the primer pair provided by the invention, wherein the final concentration of the primer is preferably 20-300 nM. Other PCR reagents may be selected according to conventional techniques, for example, a preferred embodiment further comprises a DNA polymerase, a PCR buffer, a mixture of 4 kinds of dNTPs (deoxyribose nucleotide triphosphates), and ultrapure water. Wherein the amount of DNA polymerase is 0.5-5U, and the final concentration of each dNTP is 50-500. mu.M.
The DNA polymerase can be Taq, KOD FX, etc., so that the PCR buffer solution is a concentrated buffer solution corresponding to the selected DNA polymerase, and the concentration degree can be 2X, 5X or 10X.
For example, when KOD FX is used as the DNA polymerase and 2 × concentrated buffer is used, the amounts of the components in the PCR system may be: 0.5-2 μ l of DNA polymerase, 18-30 μ l of PCR buffer solution, 1-10 μ l of mixture of various dNTPs, 1-5 μ 1 of each of upstream and downstream primers, 5-1000 ng of DNA and a proper amount of ultrapure water to make up to 50 μ l. Or preparing reaction liquid with other volume sizes according to the same proportion of the photo.
When the finished product kit is manufactured, a reagent for extracting the DNA of a sample or a professional DNA extraction kit can be selectively prepared according to actual needs; the DNA of the sample to be detected can be obtained more conveniently and quickly, and the convenience and the rapidity of the detection finished product kit are enhanced. The sample to be tested can be any blood, cell, tissue or buccal swab sample containing genomic DNA.
On the basis of the primer pair provided by the invention, the invention further provides a detection method for synchronously detecting the gene polymorphism of two SNP sites of the UGT1A1 gene, and the primer pair is adopted for PCR detection.
According to a preferred embodiment, the detection method comprises the following specific steps:
(1) extracting genome DNA from a sample to be detected as an amplification template;
(2) preparing a PCR reaction system containing the primer pair and the amplification template;
(3) carrying out PCR amplification reaction on the PCR reaction system to obtain a PCR product;
(4) and determining the gene polymorphism of two SNP sites of the UGT1A1 gene according to the PCR product.
Wherein the most preferable reaction conditions for the PCR amplification reaction are: pre-denaturation at 90-98 ℃: 1-10 min; denaturation at 90-98 ℃: 5-20 s, annealing at 58-68 ℃: 10-60 s, extension at 68-72 ℃: for 30 s-5 min, carrying out 25-40 cycles of denaturation, annealing and extension; post-extension at 67-73 ℃: hold at 2-20 ℃ for 0-30 min.
Based on the method and the conditions, the detection method can quickly, effectively and conveniently simultaneously and synchronously obtain the gene polymorphism of UGT1A1 gene of a sample to be detected at two SNP sites, particularly UGT1A1 x 28(-53TA6 > TA7) and UGT1A1 x 6(211G > A) sites of the UGT1A1 gene, and can be used for non-diagnosis purposes. In general, when UGT1A 1X 28(-53TA6 & gtTA 7) and UGT1A 1X 6(211G & gtA) sites of UGT1A1 gene were amplified using the upstream primer SEQ ID NO.1 and the downstream primer SEQ ID NO.2, the fragment length of the corresponding amplification product was 591 bp. The DNA fragment was then recovered by cutting and sequenced.
According to a preferred embodiment of the present invention, the step (4) of determining gene polymorphism comprises: electrophoretically detecting the PCR product to verify the amplified fragment size of the PCR product; and after the correctness is verified, carrying out sequence determination on the PCR product so as to obtain the gene polymorphism conditions of UGT1A1 x 28(-53TA6 > TA7) and UGT1A1 x 6(211G > A) sites of UGT1A1 gene of the sample to be detected. In detail, the PCR amplified fragment can be detected by agarose gel electrophoresis or polyacrylamide gel electrophoresis.
Compared with the prior art, the invention at least has the following beneficial effects: (1) and (3) improving the detection flux: generally, each PCR reaction only aims at one SNP site, but the invention can simultaneously detect 2 SNP sites of UGT1A1 gene, and can detect gene polymorphism of UGT1A1 x 28(-53TA6 > TA7) and UGT1A1 x 6(211G > A) sites of UGT1A1 gene by one reaction. Therefore, more than 90 samples can be detected simultaneously, the detection efficiency is improved, and the cost is greatly saved. (2) The cost is reduced: the invention can reduce the PCR reaction system from 2 systems/procedures to 1 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
FIG. 1 shows the result of agarose gel electrophoresis detection according to an embodiment of the present invention;
FIG. 2 is a diagram showing the sequencing of PCR products according to one embodiment of the present invention at site 28;
fig. 3 is a portion of the PCR product sequencing results provided in accordance with an embodiment of the present invention for the 6 site.
Detailed Description
To further illustrate the present invention, reference is made to the following examples. 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
Designing and synthesizing a primer pair, comprising the following steps:
step 1.1: based on the upstream and downstream sequences of UGT1A1 gene including UGT1A1 × 28(-53TA6 > TA7) and UGT1A1 × 6(211G > A) sites, upstream and downstream primers for specifically amplifying UGT1A1 × 28(-53TA6 > TA7) and UGT1A1 × 6(211G > A) sites are designed.
For designing primers, Primer Quest and Primer Premier 5.0 were used for Primer design and analysis of dimer and stem-loop mismatches, and primers were designed at both ends of UGT1A1 × 28(-53TA6 > TA7) and UGT1A1 × 6(211G > A) sites.
The primer pair provided in this example covers UGT1A1 × 28(-53TA6 > TA7) and UGT1A1 × 6(211G > A) sites of UGT1A1 gene. Because the amplification efficiency of the primers is obviously reduced and the specificity is deteriorated due to small sequence change, PCR primer pairs are respectively designed aiming at different sites/exons, and after the screening of a pre-experiment, the primer pairs with the best amplification effect are selected according to the length of product fragments and the inclusion conditions of the sites/exons, wherein the primer pairs are shown in the following table 1.
TABLE 1
| Primer name | Primer sequence 5 '-3' |
| SEQ ID NO.1 | CTCCCTGCTACCTTTGTGGACTGA |
| SEQ ID NO.2 | CCGTCAGCATGACATCAAAGCTGC |
Step 1.2: the primer pair designed in 1.1 was synthesized.
Example 2
The method for extracting the genome DNA from the sample to be detected comprises the following steps:
step 2.1: mouth-shed cells were collected with a mouth swab or fresh peripheral blood samples were collected with a blood collection tube.
In this embodiment, the sample source is a human body.
Step 2.2: genomic DNA was extracted from the specimen using a Tiangen buccal swab genomic DNA extraction kit (DP322) or a blood/cell/tissue genomic DNA extraction kit (DP304), and the concentration and purity of the DNA were measured using NP80-touch (IMPLEN, Germany) to preserve the genomic DNA.
Example 3
The PCR detection method for synchronously detecting UGT1A 1X 28(-53TA6 & gtTA 7) and UGT1A 1X 6(211G & gtA) locus gene polymorphism of UGT1A1 gene comprises the following steps:
step 3.1: and (3) taking the genome DNA obtained in the step 2.2 as an amplification template, and adopting the primer pair synthesized in the step 1.2 to prepare a PCR reaction system.
In this example, a PCR amplification system was prepared by using DNA polymerase and buffer solution as basic raw materials in KOD FX enzyme system (cat. KFX-101) manufactured by Toyobo, Inc., and adjusting the primer concentration, dNTP concentration, buffer solution concentration and enzyme amount based on the amplification system in the enzyme system specification, and the specific composition of this reaction system is shown in Table 2 below. Of course, the equal scale enlargement/reduction of the reaction system is within the protection scope of the embodiment of the invention; the amplification can also be achieved by replacing other DNA polymerase systems and adjusting the appropriate proportion.
TABLE 2
| Reagent composition | Volume of |
| 2×PCR buffer for FX | 20μl |
| 2mM dNTP | 8μl |
| F | 1μl |
| R | 1μl |
| KOD FX(1U/μl) | 1μl |
| DNA | 2μl |
| Water (W) | 17μl |
The primers were mixed equimolar, the primer concentration being 10 micromolar; the amount of the DNA template can be adjusted, and 20ng of genomic DNA can be used in this example.
Step 3.2: the PCR instrument was programmed according to the PCR reaction conditions shown in Table 3 below.
TABLE 3
Step 3.3: and (3) carrying out PCR amplification reaction on the PCR reaction system prepared in the step 3.1 by using a programmed PCR instrument to obtain a PCR product.
Example 4
Electrophoresis detection, comprising the following steps:
step 4.1: the PCR product obtained in step 3.3 was examined by agarose gel electrophoresis to verify the size of the PCR product fragment.
The detection results are shown in fig. 1, 1821, 1822, 1823, 1824, 1825, LSS and the like shown in fig. 1 are mainly used for distinguishing different samples to be detected, the leftmost column of fig. 1 shows a ruler bar, the rightmost column shows electrophoresis results of PCR products of a blank control group, and the middle columns show electrophoresis results of PCR products of different samples.
According to the comparison between the position of each product bright band and the left scale bar, whether the product bright band corresponds to the target amplification product can be identified.
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 each sample to be detected, the existing strip is a corresponding PCR amplification product, and the size of the strip is consistent with the theory; the strip is clear, has no smear and the like, and has good bright strip effect. Thus, when the PCR amplification primer pair designed in step 1.1 is used for PCR amplification, only the expected target product is generated, but no other unrelated product is generated, and the primer pair is reasonably designed.
Step 4.2: after the size of the PCR product fragment is verified to be correct, the sequence of the PCR product can be determined.
Example 5
Sequence determination comprising the steps of:
step 5.1: and (4) after the sizes of the PCR product fragments are verified to be correct in the step 4.2, sending the PCR product obtained in the step 3.3 to a sequencing company for sequence determination to obtain a sequencing result in the format of ab 1.
Step 5.2: the sequencing results obtained in step 5.1 were analyzed by Chromas sequence analysis software to obtain the gene polymorphism of UGT1a1 × 28(-53TA6 > TA7) and UGT1a1 × 6(211G > a) sites of UGT1a1 gene.
The partial sequencing results are shown in FIGS. 2-3.
Referring to FIG. 2, FIG. 2 shows nucleotide base sequences at and upstream and downstream of the gene mutation site UGT1A1 × 28(-53TA6 > TA 7). Referring to the box line portion in fig. 2, it can be seen that the number of TA sequence repeats at positions 89-100 is 6, i.e., the genotype at UGT1a1 × 28 is represented by × 1/× 1.
Referring to fig. 3, fig. 3 shows nucleotide base sequences at and upstream and downstream of the gene mutation site UGT1a1 × 6(211G > a). Referring to the frame line part in fig. 3, it can be seen that the base at position 356 is G, i.e., the genotype at UGT1a1 × 6 is GG type.
Example 6: preparation of the kit
According to the above experimental results, the present embodiment provides a preferred kit for simultaneously detecting gene polymorphisms at two SNP sites of UGT1a1 gene, the kit comprising the following reagents:
1. PCR primer pair: f upstream primer (10. mu.M), R downstream primer (10. mu.M);
2、KOD FX(1U/μl)；
3、2×PCR buffer for FX：
4、dNTP；
5. a sample DNA extraction reagent;
6. sample DNA collection kits (e.g., containing buccal swabs and swab kits/tubes);
7. ultrapure water.
The reagent is reasonably placed in the kit, the instructions (optionally preparing a PCR test tube or a pore plate or a pipette) of the kit are put into the kit, the instructions comprise the step of collecting a sample to be detected, the collected sample is put into a storage box/tube, the step of DNA extraction is carried out, and finally the PCR amplification process is carried out according to the detection method.
It should be noted that, in this document, 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.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.
Sequence listing
<110> Beijing and Hei medical diagnostic technology GmbH
<120> method for simultaneously detecting gene polymorphisms at two SNP sites of UGT1A1 gene
<130>ss191-212
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>24
<212>DNA
<213> Artificial sequence ()
<400>1
ctccctgcta cctttgtgga ctga 24
<210>2
<211>24
<212>DNA
<213> Artificial sequence ()
<400>2
ccgtcagcat gacatcaaag ctgc 24
Claims (10)
1. A primer pair for synchronously detecting gene polymorphism of two SNP sites of UGT1A1 gene is characterized in that the primer pair comprises a pair of primers, the nucleotide sequence of an upstream primer is shown by SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown by SEQ ID NO. 2.
2. The primer set according to claim 1, wherein the SNP sites are UGT1A 1X 28(-53TA6 > TA7) and UGT1A 1X 6(211G > A) sites of UGT1A1 gene.
3. Use of the primer pair according to claim 1 or 2 for preparing a kit for simultaneously detecting gene polymorphisms at two SNP sites of UGT1A1 gene.
4. A kit for synchronously detecting gene polymorphisms at two SNP sites of UGT1a1 gene, comprising the primer pair according to claim 1 or 2.
5. The kit according to claim 4, further comprising a DNA polymerase, a PCR buffer, a mixture of 4 dNTPs, and ultrapure water.
6. The kit according to claim 5, wherein the DNA polymerase is used in an amount of 0.5 to 5U, each dNTP is used at a final concentration of 50 to 500. mu.M, and the primer is used at a final concentration of 20 to 300 nM.
7. The kit according to any one of claims 4 to 6, further comprising a test sample DNA extraction reagent or DNA extraction kit.
8. A method for simultaneously detecting gene polymorphism of two SNP sites of UGT1A1 gene, which is characterized in that PCR detection is carried out on a sample to be detected by using the primer pair of claim 1 or 2.
9. The detection method according to claim 8, characterized by comprising the steps of:
(1) extracting genome DNA from a sample to be detected as an amplification template;
(2) preparing a PCR reaction system containing the primer pair and the amplification template;
(3) carrying out PCR amplification reaction on the PCR reaction system to obtain a PCR product;
(4) and determining the gene polymorphism of two SNP sites of the UGT1A1 gene according to the PCR product.
10. The detection method according to claim 9, wherein the reaction conditions of the PCR amplification reaction are: pre-denaturation at 90-98 ℃: 1-10 min; denaturation at 90-98 ℃: 5-20 s, annealing at 58-68 ℃: 10-60 s, extension at 68-72 ℃: for 30 s-5 min, carrying out 25-40 cycles of denaturation, annealing and extension; post-extension at 67-73 ℃: hold at 2-20 ℃ for 0-30 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010369763.9A CN111534591A (en) | 2020-04-30 | 2020-04-30 | Method for synchronously detecting gene polymorphism of two SNP sites of UGT1A1 gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010369763.9A CN111534591A (en) | 2020-04-30 | 2020-04-30 | Method for synchronously detecting gene polymorphism of two SNP sites of UGT1A1 gene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111534591A true CN111534591A (en) | 2020-08-14 |
Family
ID=71973337
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010369763.9A Pending CN111534591A (en) | 2020-04-30 | 2020-04-30 | Method for synchronously detecting gene polymorphism of two SNP sites of UGT1A1 gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111534591A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007055261A1 (en) * | 2005-11-10 | 2007-05-18 | The New Industry Research Organization | Method for determination of ugt1a1 gene polymorphism |
| CN104328181A (en) * | 2014-10-30 | 2015-02-04 | 武汉百泰基因工程有限公司 | Primer, probe, kit and detection method for detecting UGT1A1 gene polymorphism |
-
2020
- 2020-04-30 CN CN202010369763.9A patent/CN111534591A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007055261A1 (en) * | 2005-11-10 | 2007-05-18 | The New Industry Research Organization | Method for determination of ugt1a1 gene polymorphism |
| CN104328181A (en) * | 2014-10-30 | 2015-02-04 | 武汉百泰基因工程有限公司 | Primer, probe, kit and detection method for detecting UGT1A1 gene polymorphism |
Non-Patent Citations (2)
| Title |
|---|
| 张睿喆等: "UGT1A1~* 6和~*28基因多态性与伊立替康毒性关系研究", 《医药论坛杂志》 * |
| 李登等: "UGT1A1基因多态性与伊立替康致迟发性腹泻及治疗的研究进展", 《科技信息》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104805206B (en) | The kit and its detection method of detection TERT gene promoter mutation | |
| Hassawi | Allele frequency and molecular genotypes of ABO blood group system in a Jordanian population | |
| EP3438283B1 (en) | Method for detecting gene mutation | |
| US20170283856A1 (en) | Lytic composition and application thereof, kit, method for preparing nucleic acid by utilizing lytic composition, and nucleic acid analysis method | |
| WO2016167317A1 (en) | Method for detecting genetic mutation | |
| CN111394441B (en) | Primer group, kit and method for amplification of vitamin A related genes | |
| EP2522741B1 (en) | Genotyping method | |
| EP2154247A1 (en) | Method of amplifying methylated nucleic acid or unmethylated nucleic acid | |
| CN110863040A (en) | Method for detecting CYP3A5 gene polymorphism by fluorescent quantitative PCR | |
| CN110819709A (en) | Method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR (polymerase chain reaction) | |
| CN110878351A (en) | Method for detecting MTHFR gene polymorphism by fluorescent quantitative PCR | |
| EP1829979B1 (en) | Method of identifying gene with variable expression | |
| CN111534591A (en) | Method for synchronously detecting gene polymorphism of two SNP sites of UGT1A1 gene | |
| CN110904215A (en) | Method for synchronously detecting gene polymorphism of two SNP sites of SLCO1B1 gene | |
| JP6053681B2 (en) | Method and kit for diagnosing glaucoma in dogs | |
| CN112280852B (en) | SMN1 gene mutation detection kit and application thereof | |
| Hofmann et al. | Comparative analysis of hypermethylation of cell cycle control and DNA-mismatch repair genes in low-density and CD34+ bone marrow cells from patients with myelodysplastic syndrome | |
| CN111363806B (en) | Primer group for detecting vitamin B12 metabolic gene mutation and application method thereof | |
| CN110885884A (en) | Method for synchronously detecting polymorphism of CYP2C19 gene 2, 3, 17 locus gene | |
| Mömke et al. | Mapping quantitative trait loci for left-sided displacement of the abomasum in German Holstein dairy cows | |
| CN110819701A (en) | Method for detecting CYP2D6 gene polymorphism by fluorescent quantitative PCR | |
| KR101455284B1 (en) | Composition for diagnosing Moyamoya disease using CpG methylation status in promoter region of APOD gene and uses thereof | |
| CN111518888A (en) | Method for synchronously detecting gene polymorphism of two SNP sites of APOE gene | |
| CN111500707A (en) | Method for synchronously detecting polymorphism of two SNP sites of CYP3A4 gene | |
| CN111500727A (en) | Primer group for detecting KRAS gene and BRAF gene mutation and application method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200814 |
|
| RJ01 | Rejection of invention patent application after publication |