CN105039529A - Primer for detecting ERCC2 gene polymorphism, and method thereof - Google Patents
Primer for detecting ERCC2 gene polymorphism, and method thereof Download PDFInfo
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- CN105039529A CN105039529A CN201510384760.1A CN201510384760A CN105039529A CN 105039529 A CN105039529 A CN 105039529A CN 201510384760 A CN201510384760 A CN 201510384760A CN 105039529 A CN105039529 A CN 105039529A
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Abstract
The invention provides a primer for detecting the ERCC2 gene polymorphism. The primer comprises a PCR amplification primer and an SNaPshot PCR primer. The invention belongs to the technical field of biological detection. The primer provided by the invention can realize specific detection of the ERCC2 gene polymorphism, and has good accuracy.
Description
Technical field
The invention belongs to technical field of biological, particularly relate to a kind of primer and the method that detect ERCC2 gene pleiomorphism.
Background technology
Platinum-containing anticancer drug, comprising cis-platinum, carboplatin and oxaliplatin etc., is cancer therapy drug kinds cancer in current clinical application to greater activity, mainly through causing in cell DNA damage to remove cancer cells.
Excision repair cross-completion 1 gene (ERCC1), is mankind's DNA damage revision points, is positioned at karyomit(e) 19q13.2-13.3.Excision Repair Cross-Complementing Group 2 (ERCC2), participates in Nucleotide Sequence Analysis and genetic transcription, plays an important role in DNA damage reparation.
Existing data display, ERCC1_C118TC/C, C/T, T/T genotype frequency is about 52.6% respectively, and 43.1%, 4.2%; ERCC2_L751QK/K, K/Q, Q/Q genotype frequency is about 84.92% respectively, and 15.08%, 0%.Therefore, by the detection of ERCC2 gene pleiomorphism, contribute to the prognosis predicting platinum-based chemotherapy, significant in help direction of medication usage and personalized treatment.Prior art lacks primer and the method thereof that a species specificity is good, highly sensitive, can realize ERCC2 genetic polymorphism detection.
Summary of the invention
For solving problems of the prior art, the invention provides a kind of primer and the method that detect ERCC2 gene pleiomorphism, there is the advantages such as specificity is good, highly sensitive, accuracy is good, achieve the detection of ERCC2 gene pleiomorphism.The site that the present invention detects comprises ERCC2_L751Q:c.2251A>C (p.Lys751Gln) (SNP:rs13181).
The invention provides a kind of primer detecting ERCC2 gene pleiomorphism, comprise pcr amplification primer and SNaPshotPCR primer; Described pcr amplification primer comprises: for upstream primer 5 '-GGCAAGACTCAGGAGTCACC-3 ' (SEQIDNO.1) and the downstream primer 5 '-CCCTCTCCCTTTCCTCTGTT-3 ' (SEQIDNO.2) of ERCC2_L751Q; Described SNaPshotPCR primer comprises: for SNaPshotPCR the primer 5 '-TTTTTTTAGCAGCTAGAATCAGAGGAGACGCTG-3 ' (SEQIDNO.3) of ERCC2_L751Q.
Adopt technique scheme, the primer of detection ERCC2 gene pleiomorphism provided by the invention, can realize the specific detection of ERCC2 gene pleiomorphism, accuracy is good.
Correspondingly, the present invention also provides a kind of method detecting ERCC2 gene pleiomorphism, comprises the steps: A) extract DNA sample; B) adopt the pcr amplification primer described in claim 1 to carry out pcr amplification, purifying is carried out to amplified production; C) adopt the SNaPshotPCR primer described in claim 1 to carry out SNaPshotPCR amplification, purifying is carried out to amplified production; D) capillary electrophoresis technique detects, and analyzes detected result, determines SNP site and genotype thereof.
Preferably, described steps A) in DNA sample be the DNA sample of EDTA anticoagulation cirumferential blood.
Preferably, described step B) in pcr amplification reaction condition comprise: stage 1:98 DEG C 3min; Stage 2:98 DEG C 10s; Stage 3:58 DEG C 30s; Stage 4:72 DEG C 1min; Stage 5: turn back to stage 2,29 circulation; Stage 6:72 DEG C 5min; Stage 7:25 DEG C of insulation.That is, Step1:98 ° of Cfor3minutes; Step2:98 ° of Cfor10seconds; Step3:58 ° of Cfor30seconds; Step4:72 ° of Cfor1minute; Step5:Gotostep2,29times; Step6:72 ° of Cfor5minutes; Step7:25 ° of Cforever.
Described step C) in SNaPshotPCR amplification reaction condition comprise: stage 1:96 DEG C 10s; Stage 2:55 DEG C 5s; Stage 3:60 DEG C 30s; Stage 4: turn back to stage 1,25 circulation; Stage 5:4 DEG C of insulation.That is, Step1:96 ° of Cfor10seconds; Step2:55 ° of Cfor5seconds; Step3:60 ° of Cfor30seconds; Step4:Gotostep1,25times; Step5:4 ° of Cforever.
Preferably, described step D) in, adopt GENEMAPPERIDV4.1 software to analyze detected result.
In addition, the present invention also provides the purposes of the primer detecting ERCC2 gene pleiomorphism in preparation detection ERCC2 gene pleiomorphism reagent.
Compared with prior art, the invention has the beneficial effects as follows: the invention provides a kind of primer detecting ERCC2 gene pleiomorphism, the specificity of primer is good, and accuracy is good, improves detection efficiency.In addition, present invention also offers a kind of SNaPshot method detecting ERCC2 gene pleiomorphism, by using specific pcr amplification primer and SNaPshotPCR primer, there is the advantages such as specificity is good, highly sensitive, accuracy is good, guidance can be provided for the clinical application of platinum medicine.
Accompanying drawing explanation
The amplified fragments of Fig. 1 ERCC2 gene primer provided by the invention.
The part sequencer map of Fig. 2 ERCC2 gene primer amplified production.
The ERCC2 genetic polymorphism detection result figure of Fig. 3 sample.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further details.
embodiment one primer
Contriver, for the gene polymorphism sites of ERCC2, devises a large amount of primer, by the optimization of primer reaction conditions with compare, has filtered out the primer that specificity is good.Primer provided by the invention comprises pcr amplification primer and SNaPshotPCR primer, and pcr amplification primer and SNaPshotPCR primer are corresponding.All primer sequences provided by the invention all by the comparison of UCSC database, without known SNP site.
the specificity of embodiment two primer
Primer provided by the invention is carried out in UCSC Blasting, ERCC2_L751Q primer amplification fragment and be positioned at chr19:45854837+45855007, length is 171bp; As shown in Figure 1, the amplified fragments of primer all covers corresponding detection site to result, without other homologous gene.
In use table 1, pcr amplification primer increases and Sanger order-checking to detection sample respectively, and sequencing result shows, and primer amplification fragment and ERCC2 gene reference sequence coincide, and result as shown in Figure 2.Use SNaPshotPCR primer in table 1, SNaPshot method detects, and result as shown in Figure 3.As can be seen from Figure 3, the base that the relative position at each product peak and sequencing reaction mix conforms to expection, and without other Interference Peaks.
the detection of embodiment two ERCC2 gene pleiomorphism
Extract the DNA sample of EDTA anticoagulation cirumferential blood, extracting method with reference to TIANampBloodDNAKit(purchased from Tiagen, article No.: DP318) specification sheets, DNA sample is diluted to 100ng/ μ L, for subsequent use.
Pcr amplification adopts Q5
?warm start surpasses fidelity 2XMasterMix(purchased from NEB company, article No.: M0494L), reaction system is as shown in table 2, and the primer concentration of ERCC2_L751Q is 5pmol/uL.Pcr amplification reaction condition comprises: stage 1:98 DEG C 3min; Stage 2:98 DEG C 10s; Stage 3:58 DEG C 30s; Stage 4:72 DEG C 1min; Stage 5: turn back to stage 2,29 circulation; Stage 6:72 DEG C 5min; Stage 7:25 DEG C of insulation.After pcr amplification terminates, get 2.0 μ LExoSAP-IT(purchased from Affymetrix, article No.: 78205) and 3.0 μ LddH
2o, mixing, adds pcr amplification product 2.0 μ L, mixes micro-centrifugal; Endonuclease reaction is carried out, program: 37 DEG C, 15min in PCR instrument; 80 DEG C, 15min; 4 DEG C, insulation.
Adopt SNaPshot
?multiplexKit(purchased from ABI company, article No. 4323151) increase, reaction system is as shown in table 3, and the primer concentration of ERCC2_L751Q is 5pmol/uL.SNaPshotPCR reaction conditions comprises: stage 1:96 DEG C 10s; Stage 2:55 DEG C 5s; Stage 3:60 DEG C 30s; Stage 4: turn back to stage 1,25 circulation; Stage 5:4 DEG C of insulation.
In SNaPshotPCR product, add 1.0 μ LSAP enzymes, react according to following program: 37 DEG C, 60min; 75 DEG C, 15min; 4 DEG C, insulation.After completion of the reaction, capillary electrophoresis technique detects, and adopt GENEMAPPERIDV4.1 software to analyze to detected result, determine SNP site and genotype thereof, result as shown in Figure 3.
embodiment three detects the specificity of the method for ERCC2 gene pleiomorphism
The specificity of this detection method is defined as negative match-rate.Genotypic for ERCC2_L751QA/A detecting is defined as negative findings by this detection.Adopt SNaPshot method provided by the invention to detect 19 routine samples, adopt Sanger sequencing to verify simultaneously.The detected result of SNaPshot method conforms to the result of Sanger sequencing, as shown in table 4.The specificity of this detection method is 100%.
embodiment four detects sensitivity and the accuracy of the method for ERCC2 gene pleiomorphism
The sensitivity definition of this detection method is positive coincidence rate.Adopt SNaPshot method provided by the invention to detect 19 routine samples, adopt Sanger sequencing to verify simultaneously.The detected result of SNaPshot sequencing conforms to the result of Sanger sequencing, as shown in table 5.Sensitivity and the accuracy of this detection method are high.
embodiment five detects the precision of the method for ERCC2 gene pleiomorphism
The precision of this detection method is defined as carries out to sample the ability that duplicate detection obtains same result.This detection has been carried out between personnel, contrast experiment between the different hole of different time, different instrument, same sample, and result is as shown in table 6, and all results all show unanimously, and this detection precision is 100%.
Therefore, primer provided by the present invention and detection method can be used as one independently, widely used authentication method, solve the problem that ERCC2 carries out accurate Classification Identification, play the feature that PCR-SNaPshotPCR is accurate to described gene locus genotypic results, high-throughput operates, contribute to the prognosis predicting platinum-based chemotherapy, reduce untoward reaction, significant in help direction of medication usage and personalized treatment.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.
SEQUENCELISTING
<110> Guangzhou Kingmed Center for Clinical Laboratory Co., Ltd., Guangzhou medical university
<120> mono-kind detects primer and the method for ERCC2 gene pleiomorphism
<130>
<160>3
<170>PatentInversion3.3
<210>1
<211>20
<212>DNA
<213> artificial sequence
<400>ERCC2_L751Q_F
ggcaagactcaggagtcacc20
<210>2
<211>20
<212>DNA
<213> artificial sequence
<400>ERCC2_L751Q_R
ccctctccctttcctctgtt20
<210>3
<211>33
<212>DNA
<213> artificial sequence
<400>ERCC2_L751Q
tttttttagcagctagaatcagaggagacgctg33
Claims (7)
1. detect a primer for ERCC2 gene pleiomorphism, it is characterized in that: comprise pcr amplification primer and SNaPshotPCR primer; Described pcr amplification primer comprises: for upstream primer 5 '-GGCAAGACTCAGGAGTCACC-3 ' and the downstream primer 5 '-CCCTCTCCCTTTCCTCTGTT-3 ' of ERCC2_L751Q; Described SNaPshotPCR primer comprises: for SNaPshotPCR the primer 5 '-TTTTTTTAGCAGCTAGAATCAGAGGAGACGCTG-3 ' of ERCC2_L751Q.
2. detect a method for ERCC2 gene pleiomorphism, it is characterized in that: comprise the steps: A) extract DNA sample; B) adopt the pcr amplification primer described in claim 1 to carry out pcr amplification, purifying is carried out to amplified production; C) adopt the SNaPshotPCR primer described in claim 1 to carry out SNaPshotPCR amplification, purifying is carried out to amplified production; D) capillary electrophoresis technique detects, and analyzes detected result, determines SNP site and genotype thereof.
3. the method for detection ERCC2 gene pleiomorphism according to claim 2, is characterized in that: described steps A) in DNA sample be the DNA sample of EDTA anticoagulation cirumferential blood.
4. the method for detection ERCC2 gene pleiomorphism according to claim 2, is characterized in that: described step B) in pcr amplification reaction condition comprise: stage 1:98 DEG C 3min; Stage 2:98 DEG C 10s; Stage 3:58 DEG C 30s; Stage 4:72 DEG C 1min; Stage 5: turn back to stage 2,29 circulation; Stage 6:72 DEG C 5min; Stage 7:25 DEG C of insulation.
5. the method for detection ERCC2 gene pleiomorphism according to claim 2, is characterized in that: described step C) in SNaPshotPCR amplification reaction condition comprise: stage 1:96 DEG C 10s; Stage 2:55 DEG C 5s; Stage 3:60 DEG C 30s; Stage 4: turn back to stage 1,25 circulation; Stage 5:4 DEG C of insulation.
6. the method for detection ERCC2 gene pleiomorphism according to claim 2, is characterized in that: described step D) in, adopt GENEMAPPERIDV4.1 software to analyze detected result.
7. the primer of detection ERCC2 gene pleiomorphism according to claim 1 detects the purposes in ERCC2 gene pleiomorphism reagent in preparation.
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| CN201510384760.1A CN105039529A (en) | 2015-06-30 | 2015-06-30 | Primer for detecting ERCC2 gene polymorphism, and method thereof |
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- 2015-06-30 CN CN201510384760.1A patent/CN105039529A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| CHU HY等: "a genetic variant in ercc2 is associated woth gastric cancer prognosis in a chinese population", 《MUTAGENESIS》 * |
| LEE SA等: "obesity and genetic polymorphism of ercc2 and ercc4 as modifiers of risk of breast cancer", 《ECPERIMENTAL AND MOLECULAR MEDICINE》 * |
| LUCIA CORTEJOSO等: "Differential toxicity biomarkers for irinotecan- and oxaliplatin-containing chemotherapy in colorectal cancer", 《CANCER CHEMOTHER PHARMACOL》 * |
| PETER M. VALLONE: "Analyzing Single Nucleotide Polymorphisms", 《FORENSIC MITOCHONDRIAL DNA ANALYSIS: A COMMUNITY FORUM 2002 AAFS ANNUAL MEETING ATLANTA, GA》 * |
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Application publication date: 20151111 |