CN108949978A - One group is detected primer and its application of ERCC gene pleiomorphism simultaneously - Google Patents
One group is detected primer and its application of ERCC gene pleiomorphism simultaneously Download PDFInfo
- Publication number
- CN108949978A CN108949978A CN201810751036.1A CN201810751036A CN108949978A CN 108949978 A CN108949978 A CN 108949978A CN 201810751036 A CN201810751036 A CN 201810751036A CN 108949978 A CN108949978 A CN 108949978A
- Authority
- CN
- China
- Prior art keywords
- primer
- pcr
- snapshot
- amplification
- seq
- 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.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 33
- 102100031885 General transcription and DNA repair factor IIH helicase subunit XPB Human genes 0.000 claims abstract description 19
- 101000920748 Homo sapiens General transcription and DNA repair factor IIH helicase subunit XPB Proteins 0.000 claims abstract description 19
- 238000012408 PCR amplification Methods 0.000 claims abstract description 17
- 101100226017 Dictyostelium discoideum repD gene Proteins 0.000 claims abstract description 7
- 101150105460 ERCC2 gene Proteins 0.000 claims abstract description 7
- 108700031763 Xeroderma Pigmentosum Group D Proteins 0.000 claims abstract description 7
- 102100035184 General transcription and DNA repair factor IIH helicase subunit XPD Human genes 0.000 claims abstract 2
- 238000000034 method Methods 0.000 abstract description 24
- 230000003321 amplification Effects 0.000 abstract description 12
- 239000003814 drug Substances 0.000 abstract description 9
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 9
- 206010028980 Neoplasm Diseases 0.000 abstract description 6
- 201000011510 cancer Diseases 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 2
- 230000004083 survival effect Effects 0.000 abstract description 2
- 230000001225 therapeutic effect Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 24
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000012163 sequencing technique Methods 0.000 description 9
- 239000000203 mixture Substances 0.000 description 8
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 8
- 239000012634 fragment Substances 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000002512 chemotherapy Methods 0.000 description 6
- 238000007403 mPCR Methods 0.000 description 6
- 102000053196 Xeroderma Pigmentosum Group D Human genes 0.000 description 5
- 239000002585 base Substances 0.000 description 5
- 238000005251 capillar electrophoresis Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 101000876529 Homo sapiens DNA excision repair protein ERCC-1 Proteins 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000012154 double-distilled water Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 229910052697 platinum Inorganic materials 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 230000005778 DNA damage Effects 0.000 description 3
- 231100000277 DNA damage Toxicity 0.000 description 3
- 102100035186 DNA excision repair protein ERCC-1 Human genes 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 206010071602 Genetic polymorphism Diseases 0.000 description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 201000005202 lung cancer Diseases 0.000 description 3
- 208000020816 lung neoplasm Diseases 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 238000002205 phenol-chloroform extraction Methods 0.000 description 3
- 238000004393 prognosis Methods 0.000 description 3
- 238000001959 radiotherapy Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 230000010100 anticoagulation Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108010076525 DNA Repair Enzymes Proteins 0.000 description 1
- 102100033195 DNA ligase 4 Human genes 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 101150050700 ERCC1 gene Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 238000005422 blasting Methods 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 238000011337 individualized treatment Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 150000003057 platinum Chemical class 0.000 description 1
- 238000011518 platinum-based chemotherapy Methods 0.000 description 1
- 101150086745 pre gene Proteins 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
-
- 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)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses one group to detect simultaneouslyERCCThe primer of gene pleiomorphism comprising PCR amplification primer and SNaPshot PCR primer；The site of detection includes simultaneouslyERCC1 rs11615、 ERCC2 rs1799793、ERCC3 rs3738948；The primer is applied in chemotherapeutics selection, compared with routine is to the progress of multiple sites individually amplification, amplification program is not only reduced, reduces amplification cost, while ensure that highly sensitive, accuracy and precision, ensure that the reliability of this method；Facilitating doctor is that patient correctly selects drug and therapeutic scheme, improves the survival rate of cancer patient.
Description
Technical field
The invention belongs to genetic polymorphism detection fields, and in particular to one kind detects simultaneouslyERCCThe primer of gene pleiomorphism
And its application.
Background technique
Cancer death rate is only second to cardiovascular and cerebrovascular disease, based on chemotherapy of the clinical treatment means based on platinum medicine.
The action target spot of platinum medicine is DNA, and by interchain reciprocation, damage dna slows down the malignant proliferation rate of tumour cell.
Nucleotide Sequence Analysis (NER) has played important function in the injury repair approach after chemotherapeutics effect.NER is one
The excision approach of multi-functional and complicated DNA damage, by identifying that DNA damage starts.Key step during this
It is the double-spiral structure uncoiling at DNA damage site, Single-stranded DNA fragments of the excision comprising damage, between being repaired by DNA synthesis
Gap and fill up remaining single stranded gaps.Three genes ERCC1, ERCC2, the ERCC3 studied herein are in Nucleotide Sequence Analysis
It plays a significant role in approach.Wherein ERCC1 gene is considered as influencing non-small cell lung cancer platinum-based chemotherapy curative effect and pre-
Gene afterwards；ERCC2 plays despiralization；It has been reported that being carried in clinical III phase patientERCC1 rs11615
The conditions of patients remission rate of TT/TC genotype is higher than CC genotype；ERCC3 rs3738948 The conditions of patients of GG/GA genotype
Remission rate is higher than AA genotype；The toxicity of patient's chemicotherapy withERCC2 rs1799793Mononucleotide polymorphism site it is related.
Therefore, because presence heterogeneous between cancer patient itself and individual patients, same kind of tumour is to same chemotherapy side
The sensibility and toxic side effect response difference of case are very big, are examined by selectivity to such as DNA repair enzyme system gene polymorphism sites
It looks into, obtains the predictive factor of chemotherapeutic efficacy and toxic side effect, individualized treatment from now on is instructed, to improve the total prognosis of patient
And disease-free survival rate.It is assisted now clinically by third party inspection company, using the method for single-gene sequencing respectively to these
The polymorphism in site is detected；Lack that a species specificity is good, high sensitivity, accuracy is good, multiple sites can be achieved simultaneously
The technology and methods of detection.
Summary of the invention
For overcome the deficiencies in the prior art, the present invention provides one group to detect simultaneouslyERCCThe primer of gene pleiomorphism,
3 key genes i.e. for the radiotherapy and chemotherapy medicine based on platinum medicine in clinical cancer therapy in DNA reparation approach
3 polymorphic sites of ERCC1, ERCC2, ERCC3:ERCC1 rs11615、ERCC2 rs1799793、ERCC3 rs3738948, provide while detectingERCC1 rs11615、ERCC2 rs1799793 、ERCC3 rs3738948Gene is more
The primer of state property；Primer includes PCR amplification primer and SNaPshot PCR primer；
ForERCC1 rs11615 The forward primer of PCR amplification is as shown in SEQ ID NO:1, reverse primer such as SEQ ID NO:
Shown in 2, SNaPshot PCR primer is as shown in SEQ ID NO:7；
ForERCC2 rs1799793 The forward primer of PCR amplification is as shown in SEQ ID NO:3, reverse primer such as SEQ ID
Shown in NO:4, SNaPshot PCR primer is as shown in SEQ ID NO:8；
ForERCC3 rs3738948 The forward primer of PCR amplification is as shown in SEQ ID NO:5, reverse primer such as SEQ ID
Shown in NO:6, SNaPshot PCR primer is as shown in SEQ ID NO:9.
The present invention detects simultaneouslyERCC1 rs11615、ERCC2 rs1799793、ERCC3 rs3738948Gene pleiomorphism
SNaPshot detection method, first extraction cancer patient's peripheral white blood cells genomic DNA, by its concentration quantitative to 50ng/ μ
Then L is expanded by Multiplex PCRERCC1 rs11615、ERCC2rs1799793、ERCC3 rs3738948Place sequence,
Corresponding sequence is carried out SNaPshot PCR amplification after purification by amplified production, and amplified production is examined by capillary electrophoresis after purification
It surveys fluorescence signal and is analyzed by software and determine SNP site and genotype.
Above-mentioned detection method specifically comprises the following steps:
(1) DNA is extracted: leucocyte genomic DNA in peripheral blood sample is extracted using phenol chloroform method, it is dense by detecting mentioned DNA
Degree, and it is diluted to 50ng/ μ L；
(2) Multiplex PCR expandsERCC1 rs11615、ERCC2 rs1799793、ERCC3 rs3738948Place sequence, always
Include in the reaction system of 15 μ L of system: ddH2O:6.0 μ L；2 × Multiplex Buffer:7.5 μ L； Multiplex DNA
Polymerase:0.6 μ L；Primer Mix:0.3 μ L；The DNA mould of the 50ng/ μ L of 0.6 μ L is finally separately added into system
Plate.PCR reaction tube is placed in PCR instrument after mixing and response procedures are provided that 1:95 DEG C, 5min；2:95 DEG C, 30s；60
DEG C, 2min；72 DEG C, 2min；The step cycle 35 times；3:72 DEG C, 10min；4:4 DEG C；
(3) multiple PCR products purify: taking the product in 15 μ L steps (2), 5U CIAP and 2U Exol is added, after mixing
It is placed in PCR instrument, is reacted by following procedure: 1:37 DEG C, 60min；2:75 DEG C, 15min；3:4 DEG C；The product dilution of purifying arrives
50ng/μL；
(4) SNaPshot PCR amplification: SNaPshot PCR reaction system are as follows: ddH2O:2.5 μ L； 5× Sequencing
Buffer:2 μ L；Mix: 0.5 μ L of Multiplex Ready Reaction；Primer Mix:1 μ L；Purifying in step (3)
Diluted product afterwards: 4 μ L；Wherein the ratio of primer mixture is as follows: rs11615-SBE-F:rs1799793-SBE-F:
rs3738948-SBE-R=1:6:2；Above-mentioned system is mixed well micro- from merging PCR instrument execution following procedure: 1:96 DEG C,
10s；50 DEG C, 5s；60 DEG C, 30s；Circulation 25 times；2:4 DEG C；
(5) SNaPshot pcr amplification product purifies: 1U CIAP enzyme being added into SNaPshot pcr amplification product, by following
Program is reacted: 1:37 DEG C, 60min；2:75 DEG C, 15min；3:4 DEG C；
(6) SNaPshot is analyzed: the product that step 5) is obtained is examined with ABI 3500-Dx Genetic Analyser capillary electrophoresis
Fluorescence signal is surveyed, is analyzed by Genemapper5 software and determines genotype.
Above-mentioned primer another object is that is applied the choosing in chemotherapy drug and chemicotherapy therapeutic scheme by the present invention
In selecting.
One group provided by the invention is detected simultaneouslyERCC1 rs11615、ERCC2 rs1799793、ERCC3 rs3738948
The primer of gene pleiomorphism, using SNaPshot detection method, by multiplex PCR to the genetic fragment where 3 SNP sites into
Row amplification program, while being detected using SNaPshot technology, multiple sites are individually expanded and detected with conventional
Method compare, not only reduce amplification program, reduce amplification cost, while ensure that high sensitivity, accuracy and precision
Degree, ensure that the reliability of the detection method.
Detection method provided by the invention clinically can use preceding progress by non-small cell lung cancer radiotherapy and chemotherapy medicineERCC1 rs11615、ERCC2 rs1799793、ERCC3 rs3738948Genetic polymorphism detection, based on determining patient to platinum class
The sensibility of radiotherapy and chemotherapy medicine, facilitating doctor is that patient correctly selects drug, improves the prognosis of disease.
Detailed description of the invention
Fig. 1 isERCC1 rs11615、ERCC2 rs1799793、ERCC3 rs3738948Gene amplification product Sanger
Sequencer map；
Fig. 2 is the detection of SNaPshot methodERCC1 rs11615、ERCC2 rs1799793、ERCC3 rs3738948Figure.
Specific embodiment
Below by embodiment, invention is further described in detail, but protection scope of the present invention be not limited to it is described
Content, it is conventional commercial unless otherwise specified using reagent that method is all made of conventional method unless otherwise specified in embodiment
Reagent or the reagent configured using conventional method.
Embodiment 1:ERCC1 rs11615、ERCC2 rs1799793、ERCC3 rs3738948Gene pleiomorphism
SNaPshot detection
This detection method is after expanding SNP site place to be detected genetic fragment by Multiplex PCR, using fluorescent marker list alkali
Base elongation technology (SNaPshot) combines capillary electrophoresis technique to carry out genetic polymorphism detection, testing result application
GENEMAPPER software is analyzed, and the corresponding SNP site of extension products is determined according to the shift position at peak, according to the fluorescence at peak
Signal determines the genotype of the SNP site.
This detection method can be used for detecting the DNA sample of the EDTA anticoagulation cirumferential blood from patients with lung cancer, and use is multiple
After genetic fragment where PCR method expands SNP site to be detected, combined using fluorescent marker Single base extension technology (SNaPshot)
Capillary electrophoresis technique carries out genetic polymorphism detection, and testing result application GENEMAPPER software is analyzed, according to peak
Shift position determines the corresponding SNP site of extension products, and the genotype of the SNP site is determined according to the fluorescence signal at peak.
Method specifically comprises the following steps:
(1) DNA is extracted: leucocyte genomic DNA in peripheral blood sample is extracted using phenol chloroform method, it is dense by detecting mentioned DNA
Degree, and it is diluted to 50ng/ μ L；
(2) Multiplex PCR expandsERCC1 rs11615、ERCC2 rs1799793、ERCC3 rs3738948Place sequence, always
Include in the reaction system of 15 μ L of system: ddH2O:6.0 μ L；2 × Multiplex Buffer:7.5 μ L； Multiplex DNA
Polymerase:0.6 μ L；Primer Mix:0.3 μ L；The DNA mould of the 50ng/ μ L of 0.6 μ L is finally separately added into system
Plate.PCR reaction tube is placed in PCR instrument after mixing and response procedures are provided that 1:95 DEG C, 5min；2:95 DEG C, 30s；60
DEG C, 2min；72 DEG C, 2min；The step cycle 35 times；3:72 DEG C, 10min；4:4 DEG C；
(3) multiple PCR products purify: taking the product in 15 μ L steps (2), 5U CIAP and 2U Exol is added, after mixing
It is placed in PCR instrument, is reacted by following procedure: 1:37 DEG C, 60min；2:75 DEG C, 15min；3:4 DEG C；The product dilution of purifying arrives
50ng/μL；
(4) SNaPshot PCR amplification: SNaPshot PCR reaction system are as follows: ddH2O:2.5 μ L； 5× Sequencing
Buffer:2 μ L；Mix: 0.5 μ L of Multiplex Ready Reaction；Primer Mix:1 μ L；Purifying in step (3)
Diluted product afterwards: 4 μ L；Wherein the ratio of primer mixture is as follows: rs11615-SBE-F:rs1799793-SBE-F:
rs3738948-SBE-R=1:6:2；Above-mentioned system is mixed well micro- from merging PCR instrument execution following procedure: 1:96 DEG C,
10s；50 DEG C, 5s；60 DEG C, 30s；Circulation 25 times；2:4 DEG C；
(5) SNaPshot pcr amplification product purifies: 1U CIAP enzyme being added into SNaPshot pcr amplification product, by following
Program is reacted: 1:37 DEG C, 60min；2:75 DEG C, 15min；3:4 DEG C；
(6) SNaPshot is analyzed: the product that step 5) is obtained is examined with ABI 3500-Dx Genetic Analyser capillary electrophoresis
Fluorescence signal is surveyed, is analyzed by Genemapper5 software and determines genotype.
The main agents and instrument that this detection method uses are as follows:
(1) primer
This detection method the primer is by designed, designed；PCR primer sequence is shown in Table 1, and all primer sequences pass through UCSC number
It is compared according to library, without known SNP site.
1 PCR primer sequence of table
；
(2) reagent
This detection method DNA is extracted to be extracted using phenol chloroform method, and agents useful for same is that PCR amplification uses Vazyme Products
Multiplex PCR Kit, article No. PM101；The sequencing of SNaPshot Single base extension uses ABI Products SNaPshot
Multiplex Kit, article No. 4323151；Detailed step refers to respective operational manual.
(3) key instrument is shown in Table 2
Table 2 detects instrument
；
(4) primer specificity
Each primer carries out Blasting in UCSC, as a result as follows:ERCC1 rs11615Gene amplification fragment is located at chr19
45420220-45420511 length is 292bp；ERCC2 rs1799793Gene amplification fragment is located at chr19 15879539-
15879818 length are 295bp；ERCC3 rs3738948Gene amplification fragment is located at chr2 127260315-127260610 long
Spend 296bp；
3 primer amplification segment of table
Amplification is carried out to sample to be tested respectively using PCR primer in table 1 and Sanger is sequenced, sequencing result is shown, respectively expands piece
Section is coincide with corresponding site reference sequences, as a result as shown in Figure 1；Single base is carried out using SNaPshot PCR primer in table 1 to prolong
The method of stretching is detected, as a result as shown in Fig. 2, as shown in Figure 2, base that the relative position at product peak and sequencing reaction participate in and pre-
Phase is consistent, without other Interference Peaks.
Embodiment 2: the detection of specificity and sensitivity
1, specific detection
The specificity of this detection method is defined as negative match-rate, and the present invention detects 25, sample (lung cancer trouble with SNaPshot method
The DNA sample of the EDTA anticoagulation cirumferential blood of person),ERCC1 rs11615Negative rate is 32%,ERCC2 rs1799793Negative rate is
80%,ERCC3 rs3738948Negative rate is 64%.Wherein only 3 all sites are feminine gender, with sanger PCR sequencing PCR test result
Unanimously (as shown in table 4), present invention specificity are 100%.
Table 4 detection method specificity
；
2, sensitivity technique
The sensitivity definition of this detection method is positive coincidence rate, and the present invention detects 25, sample, and (EDTA of patients with lung cancer is anticoagulant
The DNA sample of peripheral blood), SNaPshot method detects that 22 sample mutation are positive, consistent with sanger PCR sequencing PCR testing result
(as shown in table 5), sensitivity of the present invention are 100%.
5 detection method sensitivity of table
3, the accuracy of detection method
It is consistent that the accuracy of this detection method is defined as distinct methods testing result；
25 sample application SNaPshot methods and the detection of Sanger PCR sequencing PCR, testing result is completely the same, as shown in table 6；This
The accuracy of detection method is 100%；
The detection of 6 accuracy of table
；
4, precision
This accuracy in detection is defined as the consistency of distinct methods testing result.
This detection carried out between personnel, different time, the comparative experiments (table 7) between same sample difference hole, all results
Display is consistent, this detection precision is 100%.
The detection of 7 precision of table
Therefore, primer provided by the present invention and combinations thereof can be used as a kind of independent, widely used identification method, solve
ERCC1 rs11615, ERCC2 rs1799793, ERCC3 rs3738948 Genotyping identify problem, play PCR-
The characteristics of SNaPshot is accurate to the gene loci genotypic results, high throughput operates helps to predict platinum medicine
The prognosis and its adverse reaction for the treatment of, it is significant to clinical cancer Chemotherapy in Patients and Radiation treatment plans selection and personalized treatment.
Sequence table
<110>Kunming University of Science and Technology
<120>one groups are detected primer and its application of ERCC gene pleiomorphism simultaneously
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial)
<400> 1
gaggtgcaag aagaggtgga 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial)
<400> 2
atgcccagag gcttctcata 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial)
<400> 3
ctctccgcag gatcaaagag 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial)
<400> 4
ccacttcacg tactccagca 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial)
<400> 5
ttctgacctg gtgctcctct 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial)
<400> 6
ccaggctgcc ttttgagata 20
<210> 7
<211> 35
<212> DNA
<213>artificial sequence (Artificial)
<400> 7
tttttttttt tttttgtact gaagttcgtg cgcaa 35
<210> 8
<211> 44
<212> DNA
<213>artificial sequence (Artificial)
<400> 8
tttttttttt tttttttttt tttttttacc ctgcagcact tcgt 44
<210> 9
<211> 60
<212> DNA
<213>artificial sequence (Artificial)
<400> 9
tttttttttt tttttttttt tttttttttt tttttttctt ttagctagag gcagtgacac 60
Claims (2)
1. one group is detected simultaneouslyERCCThe primer of gene pleiomorphism, it is characterised in that: including PCR amplification primer and SNaPshot
PCR primer；The site of detection includes simultaneouslyERCC1 rs11615、ERCC2 rs1799793、ERCC3 rs3738948；
ForERCC1 rs11615 The forward primer of PCR amplification is as shown in SEQ ID NO:1, reverse primer such as SEQ ID NO:
Shown in 2, SNaPshot PCR primer is as shown in SEQ ID NO:7；
ForERCC2 rs1799793 The forward primer of PCR amplification is as shown in SEQ ID NO:3, reverse primer such as SEQ ID
Shown in NO:4, SNaPshot PCR primer is as shown in SEQ ID NO:8；
ForERCC3 rs3738948 The forward primer of PCR amplification is as shown in SEQ ID NO:5, reverse primer such as SEQ ID
Shown in NO:6, SNaPshot PCR primer is as shown in SEQ ID NO:9.
2. described in claim 1 detect simultaneouslyERCCApplication of the primer of gene pleiomorphism in chemotherapeutics selection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810751036.1A CN108949978B (en) | 2018-07-10 | 2018-07-10 | Primers for simultaneously detecting ERCC gene polymorphism and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810751036.1A CN108949978B (en) | 2018-07-10 | 2018-07-10 | Primers for simultaneously detecting ERCC gene polymorphism and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108949978A true CN108949978A (en) | 2018-12-07 |
| CN108949978B CN108949978B (en) | 2021-10-22 |
Family
ID=64483389
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810751036.1A Active CN108949978B (en) | 2018-07-10 | 2018-07-10 | Primers for simultaneously detecting ERCC gene polymorphism and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108949978B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139631A (en) * | 2006-09-08 | 2008-03-12 | 上海主健生物工程有限公司 | Reagent case for lung carcinoma susceptibility detection through SNPs locus of XRCC1 gene and ERCC2 gene |
| US20120020984A1 (en) * | 2007-01-18 | 2012-01-26 | University Of Southern California | Genetic Markers for Predicting Responsiveness to Combination Therapy |
| CN202519261U (en) * | 2012-04-17 | 2012-11-07 | 马蓉 | ERCC1 (excision repair cross complementing 1) gene polymorphism and enzyme digestion detection kit |
| CN104988146A (en) * | 2015-06-30 | 2015-10-21 | 广州金域医学检验中心有限公司 | Primer and method for detecting ERCC1 gene polymorphism |
| CN105339507A (en) * | 2013-02-21 | 2016-02-17 | 托马生物科学公司 | Methods, compositions, and kits for nucleic acid analysis |
| US20170081724A1 (en) * | 2014-03-19 | 2017-03-23 | Dana-Farber Cancer Institute, Inc. | Method and kits for determining sensitivity to cancer treatment |
-
2018
- 2018-07-10 CN CN201810751036.1A patent/CN108949978B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139631A (en) * | 2006-09-08 | 2008-03-12 | 上海主健生物工程有限公司 | Reagent case for lung carcinoma susceptibility detection through SNPs locus of XRCC1 gene and ERCC2 gene |
| US20120020984A1 (en) * | 2007-01-18 | 2012-01-26 | University Of Southern California | Genetic Markers for Predicting Responsiveness to Combination Therapy |
| CN202519261U (en) * | 2012-04-17 | 2012-11-07 | 马蓉 | ERCC1 (excision repair cross complementing 1) gene polymorphism and enzyme digestion detection kit |
| CN105339507A (en) * | 2013-02-21 | 2016-02-17 | 托马生物科学公司 | Methods, compositions, and kits for nucleic acid analysis |
| US20170081724A1 (en) * | 2014-03-19 | 2017-03-23 | Dana-Farber Cancer Institute, Inc. | Method and kits for determining sensitivity to cancer treatment |
| CN104988146A (en) * | 2015-06-30 | 2015-10-21 | 广州金域医学检验中心有限公司 | Primer and method for detecting ERCC1 gene polymorphism |
Non-Patent Citations (6)
| Title |
|---|
| 1000GENOMES: "ss109872809", 《DBSNP DATABASE》 * |
| 1000GENOMES: "ss111425518", 《DBSNP DATABASE》 * |
| 1000GENOMES: "ss237690586", 《DBSNP DATABASE》 * |
| JEONG-SEON RYU等: "Association between polymorphisms of ERCC1 and XPD and survival in non-small-cell lung cancerpatients treated with cisplatin combinationchemotherapy", 《LUNG CANCER》 * |
| LING LI等: "Targeting DNA Damage Response in theRadio(Chemo)therapy of Non-Small Cell Lung Cancer", 《INT.J.MOL.SCI》 * |
| 肖明扬等: "核苷酸切除修复通路基因多态性与慢性苯中毒关联性", 《中国公共卫生》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108949978B (en) | 2021-10-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20080305473A1 (en) | Propagation of primary cells | |
| US20150184248A1 (en) | Method for detecting pancreatic cancer and detection kit | |
| EP2210942B1 (en) | Gene associated with liver cancer, and method for determination of the risk of acquiring liver cancer | |
| CN106978480A (en) | Molecular diagnostic assay for cancer | |
| CN105874079A (en) | Molecular diagnostic test for lung cancer | |
| WO2020048518A1 (en) | Group of genes for molecular typing of medulloblastoma and use thereof | |
| Nagahata et al. | Expression profiling to predict postoperative prognosis for estrogen receptor‐negative breast cancers by analysis of 25,344 genes on a cDNA microarray | |
| CN104988141B (en) | G.32912799T ＞ C mutation and its application in Computer-aided Diagnosis of Breast Cancer of BRCA2 genes | |
| CN109182528B (en) | Glioblastoma multiforme auxiliary diagnosis and prognosis evaluation kit based on ITGB5 gene and use method thereof | |
| Gašperšič et al. | Potential of modern circulating cell-free DNA diagnostic tools for detection of specific tumour cells in clinical practice | |
| KR102559124B1 (en) | Composition for amplifying FLT3 gene and Uses thereof | |
| Zhao et al. | Osteopontin promoter polymorphisms at locus-443 are associated with metastasis and poor prognosis of human intrahepatic cholangiocarcinoma in Chinese population | |
| CN106337081A (en) | Correlation of SNP site rs1054135 of FABP4 gene with triple-negative breast cancer prognosis | |
| CN107151707B (en) | Kit for detecting lung cancer related gene hot spot mutation and application thereof | |
| CN104630379A (en) | Non-small-cell lung cancer marker FAM107A and application thereof | |
| CN108949978A (en) | One group is detected primer and its application of ERCC gene pleiomorphism simultaneously | |
| WO2007137366A1 (en) | Diagnostic and prognostic indicators of cancer | |
| CN109439760B (en) | Application of ARRDC2 in evaluating development process of oral squamous cell carcinoma | |
| CN106811537A (en) | One kind detection epidermal growth factor receptor gene T790M low frequencies mutant primer and its application | |
| CN108342488B (en) | Kit for detecting gastric cancer | |
| CN108949977A (en) | One group is detected primer and the application of ERCC4 and XRCC1 gene pleiomorphism simultaneously | |
| Yang et al. | Mutations of METTL3 predict response to neoadjuvant chemotherapy in muscle-invasive bladder cancer | |
| CN105886609A (en) | Risk assessment of adverse drug reaction and device thereof | |
| CN110331198A (en) | It is a kind of for the SNP marker of tumor prognosis and its application | |
| US20150011411A1 (en) | Biomarkers of cancer |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |