CN105316401A - Method for measuring ABCC2 gene polymorphism - Google Patents
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Abstract
The invention belongs to the field of molecular biological techniques and medical examination, and relates to a device for detecting drug transporter ABCC2 gene polymorphism, in particular to a method for detecting ABCC2 c. 1249 G>A and ABCC 2 3972>T gene polymorphism. After DNA of a sample is extracted, conventional PCR (polymerase chain reaction) amplification (containing fluorescent dye) is carried out, and a melting curve with high resolution ratio is used for analyzing genetic typing of the sample. The method is easy and convenient to operate, a sequence specificity probe is not required, the method is not limited by basic sites, meanwhile, cross contamination is avoided by closed tube operation, and the method has the features of high efficiency, quickness, sensitivity, low cost and high flux, is suitable for clinical conventional gene detection, and further provides a technical support for clinical individual rational drug use.
Description
Technical field
The invention belongs to Protocols in Molecular Biology and field of medical examination, relate to the detection method of drug transporter ABCC2 gene pleiomorphism, be specifically related to a kind of high-resolution fusion curve analytical procedure measuring people ABCC2 gene pleiomorphism, particularly relate to a kind of method utilizing high-resolution fusion curve analytical technology to detect the gene pleiomorphism of ABCC2c.1249G>A and ABCC23972C>T.
Background technology
Prior art discloses heredity is cause one of drug reaction individual difference important factor, therefore, utilizes gene test to obtain patient's genetic information, to instructing personalized medicine, improving clinical efficacy and ensureing that drug safety is significant.The gene test product that the granted in recent years medicine of U.S. FDA is relevant increases year by year, and in original new drug specification sheets, label pharmaceutical is suitable for genotype crowd, has become the new trend of new drug development.
Drug transporters ATP is the important component part of abc transport body family in conjunction with box C subtribe (ATP-bindingcassetteC2, ABCC2), by ABCC2 genes encoding.Described ABCC2 gene is positioned at human body No. 10 chromosome long arm 24 districts (10q24), has 32 exons.Research display, there is sudden change in ABCC2 gene, found 457 single nucleotide polymorphism at present, ABCC2 gene polynorphisms can affect expression and the function of its proteins encoded; Have research to confirm, some single nucleotide polymorphism have wherein affected the activity of drug transporters and relevant with resistance; Described ABCC2c.1249G>A (rs2273697) is positioned at exons 10, its transgenation can cause the α-amino-isovaleric acid of 417 (Val) to replace to Isoleucine (Ile), transporter activity is changed, cause drug bioavailability to reduce, and improve relevant to the clearance rate of ABCC2 substrate.Also there are some researches show, ABCC2c.1249G>A saltant type increases relevant with ovarian cancer patients to the susceptibility of cis-platinum; In addition, research also finds that 417Ile sudden change is relevant to the neural system untoward reaction of Carbamzepine.ABCC23972C>T (rs3740066) is positioned at exon 28, that a same sense mutation (Ile1324Ile) may have an impact to montage, produce different transcribing, thus the albumen that translation is abnormal, the function of ABCC2 can be affected, thus cause the absorption to its substrate and discharge generation, as ABCC23972C>T polymorphism and Rinotecan metabolism and remove relevant, and affect the activity of transporter, make Patients with Non-small-cell Lung produce difference to the reaction of medicine; In addition, ABCC23972C>T is relevant with multiple antitumor drug resistance.In sum, the heritable variation detecting ABCC2 gene has more important clinical meaning to predict drug response.
At present, the detection method of gene pleiomorphism mainly contains Taqman probe method and DNA direct sequencing.Described Taqman is the quantitative PCR technique of high special, and its advantage is that step is few, not easily pollutes, and is convenient to carry out somatotype to large sample; But there is certain restriction to design of primers, the sequence near SNP be there are certain requirements, limit to by mutating alkali yl site and type; Described DNA direct sequencing is the gold standard of abrupt climatic change, but exists and waste time and energy, and the cost defect such as costly, is not suitable for and detects great amount of samples.Except above-mentioned two kinds of common methods, some gene prods are there are in market, the NimbleGenAccuSNP gene typing chips of Roche Holding Ag, utilize chip technology to carry out fast typing for the sudden change of known site, be one of hot spot application of current gene chip, described gene chip is quick, accurate, easy, laboratory report is qualitative, its shortcoming is expensive, up to hundreds of dollar, and cannot process great amount of samples simultaneously.
Based on the present situation of prior art, the present inventor intends providing a kind of efficient, sensitive, method of detecting above-mentioned single nucleotide polymorphism accurately, with further to assistance individual administration, ensure drug safety and effectively provide technical support.
Summary of the invention
The object of the invention is to the defect existed based on prior art, provide a kind of method measuring people ABCC2 gene pleiomorphism, especially a kind of high-resolution fusion curve analytical procedure of people ABCC2 gene pleiomorphism; The method can efficient, sensitive, detect ABCC2c.1249G>A (rs2273697) and ABCC23972C>T (rs3740066) gene pleiomorphism accurately.
The present invention adopts high-resolution fusion curve analysis (high-resolutionmeltinganalysis, HRM) technical measurement people ABCC2 gene pleiomorphism, the cardinal principle of described HRM is the length according to DNA sequence dna, GC content and base complementrity sex differernce, the melting curve of application of high resolution is analyzed sample, and its high temperature uniformity and temperature resolution make resolving accuracy reach differentiation to single base difference; Therefore, the method be applicable to sample sudden change, single nucleotide polymorphism, methylate, the analysis such as HLA distribution type.
Without the need to the equipment of costliness and reagent in method of the present invention, operation does not need to carry out the steps such as enzyme is cut, purifying, human contact's poisonous and harmful reagent and uv irradiating etc. can be avoided, security is better, and really achieve stopped pipe operation, reduce sample contamination, analyze and do not need probe, only in PCR reaction system, increase micro-saturated fluorescence dyestuff, cost is lower; There is easy and simple to handle, quick, highly sensitive, reproducible, low cost and other advantages, be applicable to the monitoring of conventional gene polymorphism.
Concrete, a kind of method measuring people ABCC2 gene pleiomorphism of the present invention, is characterized in that, it comprises, testing sample through extracting genome DNA, after pcr amplification, melt instrument by high resolving power and detect gene type, by high-resolution fusion curve analyzing gene polymorphism;
Pass through following steps:
(1) DNA sequence dna of goal gene is searched, for the primer pair that the mutational site design screening of goal gene is suitable;
(2) extract the genomic dna of peripheral blood cells sample, the primer utilizing step (1) to screen carries out gene amplification;
(3) detect according to the mutational site of high resolving power solubility curve method to the goal gene after amplification, represent different genotype by typical curve based on curve offset (homozygote) and curve shape change (heterozygote).
In the present invention, when HRM detects mutational site genotype, according to the preselected range of PCR response procedures, optimized gene amplification program, and melt condition preselected range optimization welding condition according to product.
In embodiments of the invention, adopt high-resolution fusion curve analyst ABCC2c.1249G>A and ABCC23972C>T gene pleiomorphism, it comprises step:
(1) DNA sequence dna of goal gene ABCC2c.1249G>A and ABCC23972C>T is searched, for the primer pair that the mutational site design screening of goal gene is suitable; Described primer pair contains the sequence that in goal gene target sequence, 18-27 bp continuous nucleotide is formed, PCR primer length 80-150bp;
ABCC2c.1249G>A (rs2273697) primer sequence
rs2273697-F：TCAATCCTTATCTTTAGGCAT
rs2273697-R：ACAGTATCGCATTAATTGGG
ABCC23972C>T (rs3740066) primer sequence
rs3740066-F：CAAATGATGAAGGCTTAGGG
rs3740066-R：CTGGGTGACTGATAAGAGGC
(2) extract the genomic dna of peripheral blood cells sample, carry out gene amplification with the primer that step (1) is screened: carry out in 20 μ LPCR reaction systems, this reaction system comprises primer, archaeal dna polymerase, dNTPs, MgCl
2, template DNA, PCR damping fluid and fluorescence dye; TaKaRaPremixTaq test kit is selected to comprise Taq
tMhS, PCR damping fluid is (containing Mg
2+), dNTPs, reaction system final concentration consists of:
Described dNTPs mixed solution comprises the mixed solution of dATP, dTTP, dGTP, dCTP;
The condition of pcr amplification reaction is 92-97 DEG C of denaturation 3-15min, 92-97 DEG C of sex change 10-30s, 50-65 DEG C of annealing 10-30s, and 72 DEG C extend 10-30s, 70-75 DEG C and continue to extend 3-10min, 30-50 circulation; Analyze whether PCR primer is specific amplification through agarose gel electrophoresis method;
(3) detect according to the mutational site of high resolving power solubility curve method to the goal gene after amplification, select SYTO-9 saturated fluorescence dyestuff, application Rotor-GeneQ software analysis HRM result, different genotype is represented based on curve offset (homozygote) and curve shape change (heterozygote) by typical curve, adopt the genotypic sample DNA in three kinds, known ABCC2c.1249G>A and ABCC23972C>T site for contrast, with reference to the genotypic judgement of the check sample complete paired samples of HRM curve;
Described condition of carrying out mutational site detection is 92-97 DEG C of sex change 2-5min, 40 DEG C of renaturation 1-2min; Then initial melting temperature (Tm) 60-65 DEG C of start program heats up and melts to 95 DEG C, and detects fluorescent signal in real time in fusion processes, and 25-45 time per second; Then 40 DEG C are carried out cooling 5-15s;
The each volume components of described PCR reaction system is:
20 μ LPCR reaction systems are as follows:
Described melting curve temperature setting range is respectively: ABCC2c.1249G>A is 79-86 DEG C and ABCC23972C>T is 83-90 DEG C.
Analytical procedure method of the present invention also has the following advantages:
1) high-throughput: detect for 1 time and can complete 384 samples mensuration;
2) highly sensitive: without the need to sequence-specific probes in test, without the need to order-checking, be not also subject to the limitation of mutating alkali yl site and type;
3) specificity is high: stopped pipe operates, and avoids polluting the false positive caused;
4) cost is low: agents useful for same price is inexpensive, and cost of labor is minimum.
5) fast and convenient: in 60-90 minute, to complete detection, fast, easy and simple to handle, the applicable routine clinical monitoring of detection speed;
Analytical procedure of the present invention utilizes HRM method to detect the gene type in mutational site, be not subject to the limitation of mutating alkali yl site and type, with reference to control curve, the genotypic judgement of sample can be completed, highly sensitive, specificity is high, quick and precisely, easy and simple to handle, cost is low, thus provide genetic information, for rational use of drug and clinical drug monitoring are offered help for clinical individual medication.
Accompanying drawing explanation
Fig. 1 is the electrophorogram that embodiment of the present invention 1ABCC2c.1249G>A site DNA carries out pcr amplification and obtains under different annealing temperature.
Fig. 2 is the electrophorogram that embodiment of the present invention 2ABCC23972C>T site DNA carries out pcr amplification and obtains under different annealing temperature.
Fig. 3 is that the high resolution in the embodiment of the present invention 1 large sample ABCC2c.1249G>A site melts canonical plotting.
Fig. 4 is that the high resolution in the embodiment of the present invention 2 large sample ABCC23972C>T site melts canonical plotting.
Fig. 5 is that the high resolution in the embodiment of the present invention 1 large sample ABCC2c.1249G>A site melts difference curve figure.
Fig. 6 is that the high resolution in the embodiment of the present invention 2 large sample ABCC23972C>T site melts difference curve figure.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described further:
Embodiment 1: utilize high-resolution fusion curve analytical technology to detect the gene pleiomorphism of ABCC2c.1249G>A (rs2273697)
1. design primer:
The sequence information of people ABCC2c.1249G>A (rs2273697) gene is retrieved from the nucleic acid database of US National Biotechnology Information center, suitable primer pair is screened by PrimerPremier5 (Premier, Canada) software design; Described primer pair contains the sequence that in goal gene target sequence, 18-27 bp continuous nucleotide is formed, PCR primer length 80-150bp.
The sequence of described ABCC2c.1249G>A (rs2273697) gene:
rs2273697-F：TCAATCCTTATCTTTAGGCAT
rs2273697-R：ACAGTATCGCATTAATTGGG
2. extract the genomic dna of peripheral blood cells sample, carry out gene amplification;
TaKaRaPremix solution is selected in test, adds DNA profiling and two primers in solution, adds pure water and supplies PCR reaction system and react; In reaction system, add micro-SYTO-9 dyestuff simultaneously, reduce open pipe in subsequent operations, avoid the pollution between sample;
20 μ LPCR reaction systems are as follows:
Analyze whether PCR primer is specific amplification through agarose gel electrophoresis method, ABCC2c.1249G>A (rs2273697) site DNA carries out the electrophorogram (as shown in Figure 1) that pcr amplification obtains under different annealing temperature; ABCC2c.1249G>A (rs2273697) pcr amplification reaction condition is: 95 DEG C of denaturation 3min, 95 DEG C of sex change 20s, 55 DEG C of annealing 20s, and 72 DEG C extend 20s, and 72 DEG C are continued to extend 5min, 40 circulations;
3. according to high resolving power solubility curve method, the goal gene mutational site after amplification is detected: the optimization of HRM condition mainly the starting temperature of melting temperature (Tm), monitor fluorescence p.s. number of times two in be optimized adjustment, obtain the high resolving power melting curve figure (as shown in Figure 3) of ABCC2c.1249G>A (rs2273697) crt gene type; It is as shown in table 1 that product melts condition:
Table 1.ABCC2c.1249G>A (rs2273697) PCR primer melts condition setup program
Result shows, in the DNA detection that 10 Patients with Peripheral blood extract, 5 routine ABCC2c.1249G>A loci gene types are had to be GG type, 4 routine genotype are AG type, 1 routine genotype is AA type, show that present method has highly sensitive, that specificity good, resolving power is high feature, be applicable to ABCC2c.1249G>A genotype detection.
Embodiment 2: utilize high-resolution fusion curve analytical technology to detect the gene pleiomorphism of ABCC23972C>T (rs3740066)
1. design primer:
The sequence information of people ABCC23972C>T (rs3740066) gene is retrieved from the nucleic acid database of US National Biotechnology Information center, suitable primer pair is screened by PrimerPremier5 (Premier, Canada) software design; Described primer pair contains the sequence that in goal gene target sequence, 18-27 bp continuous nucleotide is formed, PCR primer length 80-150bp;
The sequence of described ABCC23972C>T (rs3740066) gene is:
rs3740066-F：CAAATGATGAAGGCTTAGGG
rs3740066-R：CTGGGTGACTGATAAGAGGC
2. extract the genomic dna of peripheral blood cells sample, carry out gene amplification;
Select TaKaRaPremix solution, in solution, add DNA profiling and two primers, add pure water and supply PCR reaction system and react; In reaction system, add micro-SYTO-9 dyestuff simultaneously, reduce open pipe in subsequent operations, avoid the pollution between sample;
20 μ LPCR reaction systems are as follows:
Analyze whether PCR primer is specific amplification through agarose gel electrophoresis method; ABCC23972C>T gene locus DNA carries out the electrophorogram (as shown in Figure 2) that pcr amplification obtains under different annealing temperature; The pcr amplification reaction condition of the ABCC23972C>T gene optimized is: 95 DEG C of denaturation 3min, 95 DEG C of sex change 20s, 55 DEG C of annealing 20s, and 72 DEG C extend 20s, and 72 DEG C are continued to extend 5min, 40 circulations;
3. according to high resolving power solubility curve method, the goal gene mutational site after amplification is detected: the optimization of HRM condition mainly the starting temperature of melting temperature (Tm), monitor fluorescence p.s. number of times two in be optimized adjustment, obtain the high resolving power melting curve figure (as shown in Figure 4) of ABCC23972C>T crt gene type; It is as shown in table 2 that the product optimized melts condition:
Table 2.ABCC23972C>T (rs3740066) PCR primer melts condition setup program
Result shows, in the DNA detection that 10 Patients with Peripheral blood extract, 4 routine ABCC23972C>T loci gene types are had to be CC type, 5 routine genotype are CT type, 1 routine genotype is TT type, show that present method has highly sensitive, that specificity good, resolving power is high feature, be applicable to ABCC23972C>T genotype detection.
SEQUENCELISTING
<110> Huashan Hospital Affiliated To Fudan Univ
<120> mono-kind measures the method for people ABCC2 gene pleiomorphism
<130>20141231
<160>4
<170>PatentInversion3.3
<210>1
<211>21
<212>DNA
<213>ABCC2c.1249G>A(rs2273697)--rs2273697-F
<400>1
tcaatccttatctttaggcat21
<210>2
<211>20
<212>DNA
<213>ABCC2c.1249G>A(rs2273697)--rs2273697-R
<400>2
acagtatcgcattaattggg20
<210>3
<211>20
<212>DNA
<213>ABCC23972C>T(rs3740066)--rs3740066-F
<400>3
caaatgatgaaggcttaggg20
<210>4
<211>20
<212>DNA
<213>ABCC23972C>T(rs3740066)--rs3740066-R
<400>4
ctgggtgactgataagaggc20
Claims (7)
1. measure a method for people ABCC2 gene pleiomorphism, it is characterized in that, it comprises, and testing sample, through extracting genome DNA, after pcr amplification, melts instrument by high resolving power and detects gene type, by high-resolution fusion curve analyzing gene polymorphism;
Pass through following steps:
(1) DNA sequence dna of goal gene is searched, for the primer pair that the mutational site design screening of goal gene is suitable;
(2) extract the genomic dna of peripheral blood cells sample, the primer utilizing step (1) to screen carries out gene amplification;
(3) detect according to the mutational site of high resolving power solubility curve method to the goal gene after amplification, represent different genotype by typical curve based on curve offset (homozygote) and curve shape change (heterozygote).
2., by the method for mensuration people ABCC2 gene pleiomorphism according to claim 1, it is characterized in that,
High-resolution fusion curve analyst ABCC2c.1249G>A and ABCC23972C>T gene pleiomorphism is adopted by following steps:
(1) DNA sequence dna of goal gene ABCC2c.1249G>A and ABCC23972C>T is searched, for the primer pair that the mutational site design screening of goal gene is suitable;
(2) extract the genomic dna of peripheral blood cells sample, the primer utilizing above-mentioned steps (1) to screen carries out gene amplification;
The condition of pcr amplification reaction is 92-97 DEG C of denaturation 3-15min, 92-97 DEG C of sex change 10-30s, 50-65 DEG C of annealing 10-30s, and 72 DEG C extend 10-30s, 70-75 DEG C and continue to extend 3-10min, 30-50 circulation; Agarose gel electrophoresis method is adopted to analyze whether PCR primer is specific amplification after gene amplification;
(3) detect according to the mutational site of high resolving power solubility curve method to the goal gene after amplification, select SYTO-9 saturated fluorescence dyestuff, application Rotor-GeneQ software analysis HRM result, represents different genotype by typical curve based on curve offset (homozygote) and curve shape change (heterozygote);
Described condition of carrying out mutational site detection is 92-97 DEG C of sex change 2-5min, 40 DEG C of renaturation 1-2min; Then initial melting temperature (Tm) 60-65 DEG C of start program heats up and melts to 95 DEG C, and detects fluorescent signal in real time in fusion processes, and 25-45 time per second; Then 40 DEG C are carried out cooling 5-15s.
3., by the method for mensuration people ABCC2 gene pleiomorphism according to claim 2, it is characterized in that, in described step (1), primer pair contains the sequence that in goal gene target sequence, 18-27 bp continuous nucleotide is formed, PCR primer length 80-150bp;
Described ABCC2c.1249G>A (rs2273697) primer sequence is as shown in SEQ.IDNO1. and SEQ.IDNO2.;
Described ABCC23972C>T (rs3740066) primer sequence is as shown in SEQ.IDNO3. and SEQ.IDNO4..
4., by the method for mensuration people ABCC2 gene pleiomorphism according to claim 2, it is characterized in that, in described step (2), gene amplification is carried out in 20 μ LPCR reaction systems, and this reaction system comprises primer, archaeal dna polymerase, dNTPs, MgCl
2, template DNA, PCR damping fluid and fluorescence dye; TaKaRaPremixTaq test kit is selected to comprise Taq
tMhS, PCR damping fluid is (containing Mg
2+), dNTPs, reaction system final concentration consists of:
5., by the method for mensuration people ABCC2 gene pleiomorphism according to claim 4, it is characterized in that, described dNTPs mixed solution comprises the mixed solution of dATP, dTTP, dGTP, dCTP.
6., by the high-resolution fusion curve analytical procedure of mensuration people ABCC2 gene pleiomorphism according to claim 2, it is characterized in that, each volume components of described PCR reaction system:
20 μ LPCR reaction systems are as follows:
。
7. by the method for mensuration people ABCC2 gene pleiomorphism according to claim 2, it is characterized in that, described melting curve temperature setting range difference: ABCC2c.1249G>A is 79-86 DEG C, ABCC23972C>T is 83-90 DEG C.
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| CN107090499A (en) * | 2017-04-21 | 2017-08-25 | 云南仁桥医疗科技有限公司 | A kind of kit of detection CYP21 gene mutations |
| CN109576363A (en) * | 2019-02-11 | 2019-04-05 | 中日友好医院 | A kind of primer design method and kit of people ABCC2 genetic polymorphism detection |
| CN110106236A (en) * | 2019-05-06 | 2019-08-09 | 上海派森诺生物科技股份有限公司 | Utilize the kit and its method of high-resolution melting curve method detection Levetiracetam medication related gene SCN1A polymorphism |
| CN110846387A (en) * | 2019-11-29 | 2020-02-28 | 北京艾迪康医学检验实验室有限公司 | Primer and method for detecting newborn diabetes related gene ABCC8 c.1686C > CT site mutation |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107090499A (en) * | 2017-04-21 | 2017-08-25 | 云南仁桥医疗科技有限公司 | A kind of kit of detection CYP21 gene mutations |
| CN109576363A (en) * | 2019-02-11 | 2019-04-05 | 中日友好医院 | A kind of primer design method and kit of people ABCC2 genetic polymorphism detection |
| CN109576363B (en) * | 2019-02-11 | 2022-04-29 | 中日友好医院 | Primer design method and kit for detecting polymorphism of human ABCC2 gene |
| CN110106236A (en) * | 2019-05-06 | 2019-08-09 | 上海派森诺生物科技股份有限公司 | Utilize the kit and its method of high-resolution melting curve method detection Levetiracetam medication related gene SCN1A polymorphism |
| CN110846387A (en) * | 2019-11-29 | 2020-02-28 | 北京艾迪康医学检验实验室有限公司 | Primer and method for detecting newborn diabetes related gene ABCC8 c.1686C > CT site mutation |
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Application publication date: 20160210 |