CN101591697A - The genotypic test kit of a kind of detection purinethol methyl transferase gene A719G pleomorphism site, method and purposes - Google Patents
The genotypic test kit of a kind of detection purinethol methyl transferase gene A719G pleomorphism site, method and purposes Download PDFInfo
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- CN101591697A CN101591697A CNA2008101130878A CN200810113087A CN101591697A CN 101591697 A CN101591697 A CN 101591697A CN A2008101130878 A CNA2008101130878 A CN A2008101130878A CN 200810113087 A CN200810113087 A CN 200810113087A CN 101591697 A CN101591697 A CN 101591697A
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Abstract
The invention belongs to the gene engineering field, relate to the genotypic test kit of a kind of detection purinethol methyl transferase gene A719G pleomorphism site, method, purposes.The present invention adopts the single nucleotide polymorphism methods of genotyping of quantitative fluorescent PCR, Auele Specific Primer and fluorescently-labeled probe that this method is used purinethol methyl transferase gene pleomorphism site carry out the polymerase chain reaction to sample DNA, carry out fluorescent scanning subsequently, carry out genotypic classification according to fluorescence types, wherein primer comprises the Auele Specific Primer that detects purinethol methyltransgerase detection A719G pleomorphism site.Probe comprises the wild-type and the mutant specific probe of purinethol methyltransgerase A719G pleomorphism site.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of purinethol methyl transferase gene type detection kit, method and purposes.More specifically, the present invention relates to, the genotypic test kit of a kind of analysis of encoding purinethol methyl transferase gene A719G pleomorphism site, method and purposes are provided by polymerase chain reaction method.
Background technology
Purine medicaments, comprise Ismipur (6-MP), 6-thioguanine (6-TG) and mercapto azoles purine (AZA) are clinical tumor chemotherapeutic drugs commonly used, and they comprise that by disturbing intravital nucleic acid metabolism the de novo synthesis of nucleic acid and salvage route play a role.Purine medicaments is no inherent bioactive medicine, must be by competence exertion leukemia effect after a series of metabolism in the body.
Studies show that, and the curative effect of purine medicaments and purinethol methyltransgerase (thiopurine methyltransferase, TPMT) active closely related, clinical study shows, 6-MP, the clinical efficacy of 6-TG and AZA and untoward reaction are active relevant with TPMT.The active high individuality of TPMT, the concentration that generates active metabolite-thioguanine phosphoric acid salt (TGNs) after the medication is low, so the recurrence rate height of disease; And for the individuality of TPMT defective, the purine medicaments of medical routine dose also can cause the phosphatic accumulation of thioguanine in the body, produce serious toxic side effect such as serious bone marrow depression and hepatic injury etc., being forced to of finally causing treating interrupted, and causes failure (Coulthard SA, the Howell C of treatment, Robson J, et al.Blood, 1998,92:2856-2862; Relling MV, Hancock ML, Boyett JM, et al.Blood, 1999,93:2817-2823).
Human TPMT gene length overall is 3.4kb, and encoding sequence length overall 2.7kb, open reading frame contain 735 bases.The molecular weight of the TPMT of coding is approximately 30000.Form by 245 amino-acid residues.Find that after deliberation the TPMT gene has 3 important sites, is respectively the 238th site in the nucleotide sequence, the 460th site and the 719th site.When undergoing mutation in three sites, i.e. the 238th guanine (G) → cytosine(Cyt) (C), the result makes Ala (the L-Ala) → Pro (proline(Pro)) of the 80th of aminoacid sequence, the 460th guanine (G) → VITAMIN B4 (A), make Ala (the L-Ala) → Thr (Threonine) of the 154th of aminoacid sequence, the 719th VITAMIN B4 (A) → guanine (G) makes Tyr (tyrosine) → Cys (halfcystine) of the 240th.The attenuating or the forfeiture of sudden change inductive TPMT enzymic activity, cause when using purine medicaments, forming the TGNs of high density, cause tangible hemopoietic tissue cytotoxicity, produce serious consequence, even cause death (Tai HL, Krynetski EY, Yates CR, et al.Am J Hum Genet, 1996,58:694-702; KrynetskiE Y, Schuetz JD, Galpin AJ, et al.Proc Natl Acad Sci USA, 1995,92:949).
Discover, between the genotype of TPMT and its enzymic activity very strong dependency is arranged, according to TPMT gene pleiomorphism and activity relationship, can be by detecting the genotype of patient TPMT, infer the height of TPMT enzymic activity in patient's body, predict curative effect and the side effect of different patients, the reference when providing medication for the doctor for purine medicaments.
According to bibliographical information both domestic and external, the method that detects the TPMT gene pleiomorphism mainly contains analytical procedures such as fluorescence quantifying PCR method, allele-specific PCR (allele specific PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), dhplc analysis (DHPLC), SNaP shot fixed point sequential analysis detection and gene sequencing.
Summary of the invention
The purpose of this invention is to provide a kind of reliable and stable genotypic test kit of high-throughout detection purinethol methyl transferase gene A719G pleomorphism site, method and purposes.
For achieving the above object, the present invention is by the following technical solutions:
Use purinethol methyl transferase gene genotypic Auele Specific Primer of A719G pleomorphism site and probe, sample DNA is carried out the polymerase chain reaction, carry out the scanning of fluorescence labeling probe fluorescent signal simultaneously and judge the genotypic test kit of genotypic a kind of detection purinethol methyl transferase gene A719G pleomorphism site, this test kit comprises gene-specific primer and the probe that detects purinethol methyltransgerase A719G pleomorphism site.
Wherein, the forward primer of detection purinethol methyl transferase gene is: 5 '-CCTCAAAAACATGTCAGTGTGATTT-3; Reverse primer is: 5 '-CCTGATGTCATTCTTCATAGTATTTTAACA-3 '.
The wild-type probe that detects purinethol methyl transferase gene A719G pleomorphism site is: VIC-TGTAAGTAGATATAACTTTTC-MGB; The mutant probe is: FAM-TAAGTAGACATAACTTTTCA-MGB.
Test kit provided by the invention can also contain PCR such as PCR reaction mixture and Taq archaeal dna polymerase and use reagent.Wherein, the PCR reaction mixture contains 100mM Tris-HCl, 100mM KCl, 5.0uM MgCl
2, 0.4mMdATP, 0.4mM dCTP, 0.4mM dGTP, 0.4mM dTTP; The content of Taq archaeal dna polymerase is 1-10U/ul, preferred 5U/ul.
Test kit provided by the invention can also contain the wild-type of purinethol methyl transferase gene A719G pleomorphism site and the positive control of mutant.Positive control is selected from genomic dna, RNA or is cloned into dna fragmentation on the carrier, and wherein carrier is a plasmid vector.
Simultaneously, the present invention also provides the genotypic test kit of applying detection purinethol methyl transferase gene A719G pleomorphism site to detect the method for purinethol methyl transferase gene A719G pleomorphism site.Wherein, the gene amplification program of detection purinethol methyl transferase gene A719G pleomorphism site is:
95 ℃ of pre-sex change 10 minutes; 95 ℃ of sex change 15 seconds, 58 ℃ of annealing 15 seconds, 60 ℃ were extended 45 circulations 45 seconds.
Simultaneously, the present invention provides again and detects the genotypic test kit of purinethol methyl transferase gene A719G pleomorphism site in the curative effect of prediction purine medicaments and the purposes in the side effect.Wherein, purine medicaments is Ismipur, 6-thioguanine or mercapto azoles purine.Concrete, when purinethol methyl transferase gene A719G pleomorphism site genotype is a wild-type, enzymic activity is unaffected, can use the purine medicaments of routine dose.Genotype is the homozygous mutation type, can cause the forfeiture of TPMT enzymic activity, if with routine dose medicine can cause severe side effect, thereby this type of pharmacological agent is not used in suggestion.The genotype of heterozygosis can cause the reduction of enzymic activity, and suggestion reduces the dosage of medicine.
The present invention detects the genotypic step of sample:
1. sample process
2., then need to measure the concentration of sample, and concentration of specimens is adjusted to suitable scope if sample is a dna sample.If sample is RNA then needs to transcribe and become cDNA.
3. dispose the PCR reaction mixture: PCR reaction mixture: 2.5ul;
Primer+probe: 1ul;
H2O：6.5ul；
Amount to: 10ul.
4.TPMT the amplification of gene fragment
The PCR response procedures is as follows
95 ℃ of pre-sex change 10 minutes; 95 ℃ of sex change 15 seconds, 58 ℃ of annealing 15 seconds, 60 ℃ were extended 45 circulations 45 seconds.
5. the fluorescent scanning of probe
On ABI 7900 instruments or similar instrument, scan fluorescence types, use SDS software analysis scanning result.
6. the sample genotype is judged
Karyomit(e) among the human cell exists in pairs, and in paired karyomit(e), the chromosomal genotype of each bar may be identical or different.According to this situation, each genotype can be divided into homozygous wildtype again, heterozygous and homozygous mutation type.When two karyomit(e)s in TPMT 719 sites were A, genotype was a wild-type; A karyomit(e) when TPMT 719 sites is G, and when a karyomit(e) was A, genotype was a heterozygous; When two karyomit(e)s in TPMT 719 sites were G, genotype was the homozygous mutation type.
7. sample enzymic activity prediction to be checked and purine medicaments outcome prediction
The wild gene type, enzymic activity is unaffected, can use the purine medicaments of routine dose.Genotype is the homozygous mutation type, can cause the forfeiture of TPMT enzymic activity, if with routine dose medicine can cause severe side effect, thereby this type of pharmacological agent is not used in suggestion.The genotype of heterozygosis can cause the reduction of enzymic activity, and suggestion reduces the dosage of medicine.
Biological sample of the present invention is selected from dna sample, the oligonucleotide sample of RNA sample and synthetic.
Test kit of the present invention is measured the needed specific primer of above-mentioned pleomorphism site except comprising, the conventional assembly of the test kit that also comprises the utilization pcr amplification and detect, reagent, damping fluid etc., those skilled in the art are familiar with these conventional assembly and detection methods.
Test kit provided by the invention can contain particularly:
1, PCR reaction reagent specifically comprises:
(1) primer comprises that at purinethol methyl transferase gene gene A genotypic forward primer of 719G pleomorphism site and reverse primer, they can increase and contain the gene segment in the specific mutational site of purinethol methyl transferase gene;
(2) 2 * PCR reaction mixtures: 1.0ml[100mM Tris-HCl, 100mM KCl, pH8.3 (20 ℃)]; 5.0uMMgCl
2Each 0.4mM dATP, dCTP, dGTP, dTTP, the aseptic double-distilled water preparation, pH 7.0;
(3) Taq archaeal dna polymerase (5U/ul); The Taq enzyme is preserved damping fluid: 20mM Tris-HCl (pH 8.0), 100mMKCl, 0.1mM EDTA, 1mM DTT, 0.5%NP40,0.5% (v/v), Tween 20,50% glycerine;
(4)dNTP
(5)Mg
2+
(6) PCR water: the distilled water of sterilization or water for injection.
2, specific fluorescently-labeled probe
The mark fluorescent group of probe 5 ' end can be that intermediary such as FAM, ROX, TET, HEX, TAMRA, CY3, CY5, JOE, Texas red are a kind of, and the labelling groups of 3 ' end can be TAMRA, a kind of among the NFQ.
3, standard substance comprise positive control, promptly contain the wild plasmid DNA and the mutant plasmid DNA of TPMT gene A 719G pleomorphism site.Negative control: through the distilled water of DNAase I processing.
The genotype detection test kit that utilizes Taqman real-time quantitative PCR technology provided by the invention increases substantially the amplification efficiency of PCR reaction, and the pcr template of 2ng can access the PCR product of about 2ug when reaction finishes, can satisfy the needs of detection fully.We have designed primer according to the particular case of TPMT gene, and through groping, detect each sample and can save 3 hours time.
In the present invention, we have made up and have contained the genotypic positive control plasmid of TPMTA719G pleomorphism site.
For A719G site base is the gene fragment of A (wild-type), we utilize the A719G Auele Specific Primer, it is the gene fragment of A (wild-type) that amplification obtains A719G site base, determine after the gene order of amplified fragments through the gene sequencing method, connect in the T carrier, through transformed into escherichia coli competent cell DH5 α, screening obtains positive colony, after extracting plasmid, by enzyme cut, the mode of PCR and sequencing verifies that all prompting obtains correct positive plasmid.With this positive colony large scale culturing, therefrom extract plasmid and carry out purifying, cryopreservation after mensuration concentration and the purity promptly obtains the positive control plasmid that test kit uses.
For A719G site base is the gene fragment of G (mutant), because the ratio that mutant accounts in the crowd is less, clone gene need detect very big sample size from genome, therefore adopts the mode of synthetic gene.According to gene order, synthetic contain the gene order in TPMT 719G site, be cloned among the vector plasmid pUC57, after the sequencing checking, obtain positive to plasmid.
The wild gene type, enzymic activity is unaffected, can use the purine medicaments of routine dose.Genotype is the homozygous mutation type, can cause the forfeiture of TPMT enzymic activity, if with routine dose medicine can cause severe side effect, thereby this type of pharmacological agent is not used in suggestion.The genotype of heterozygosis can cause the reduction of enzymic activity, and suggestion reduces the dosage of medicine.
The invention has the beneficial effects as follows:
(1) the present invention designs specific pcr amplification primer at A719G (Tyr240 Cys) the pleomorphism site genotype of purinethol methyltransgerase TPMT especially, and amplification efficiency height, specificity be good, save time, and better use value is arranged.
(2) the present invention designs the probe of specific fluorescent mark especially at A719G (Tyr240 Cys) the pleomorphism site genotype of purinethol methyltransgerase TPMT, the specificity height, and false positive rate is low.
(3) in the present invention, the sample genotype that we obtain detection through the sequencing checking after, be that the gene fragment amplification of A (wild-type) comes out with A719G site base, be cloned in the carrier, obtain to contain the positive control plasmid of 719A wild-type sequence; In addition, synthetic contain the gene order in TPMT 719G site, the mutant fragment of the above-mentioned 719G of containing is built into the positive control plasmid that contains 719G mutant sequence, and once more through the sequencing conclusive evidence.Such positive reference substance sequence is clear and definite, can unconfinedly increase, and obtains a large amount of positive controls, and the mass ratio of easier control reference substance such as concentration and purity.The convenient acquisition is easy to produce in enormous quantities, is convenient to controlling quality again.
(4) working method of the present invention is simple, and the technician who is familiar with general molecular biology operation can finish whole process through simple training.Detected result is applicable to extensive high-throughout pattern detection needs accurately and reliably.
(5) the present invention learns index in conjunction with individual other biological and predicts for the curative effect and the side effect of purine medicaments according to the gene pleiomorphism result of TPMT.Detected result provided by the invention makes the doctor reduce the blindness of selection of clinical purine medicaments, on the basis of polymorphism gene type assay, can predict validity and the security of using purine medicaments, instruct clinical application, to carry out the individuation medical treatment, reduce toxic side effect, reduce medical treatment cost.
Description of drawings
Fig. 1 is the sample P CR result in the TPMTA719G wild-type positive control plasmid construction process
M:100bp Ladder; 1: sample 1PCR result; 2: sample 2PCR result; 3: sample 3PCR result; 4: sample 4PCR result
Fig. 2 is the result of 20 patient A719G fragment PCR amplification back fluorescent scannings.
The invention will be further described below in conjunction with embodiment, and all this areas of having done according to the disclosure of invention are equal to replacement, all belong to protection scope of the present invention.
Embodiment
Embodiment 1
Be that embodiment does further the present invention and elaborates with TPMTA719G wild-type positive control plasmid below.
1. primer and template
Gene fragment amplification uses following primer:
TPMT?719?For：5’-CCTCAAAAACATGTCAGTGTGATTT-3；
TPMT719?Back：5’-CCTGATGTCATTCTTCATAGTATTTTAACA-3’。
Template adopts human genome DNA (numbering AI011611, concentration 15ng/ μ l)
2. gene fragment amplification
Configuration pcr amplification mixture:
Template (1515ng/ μ l): 3 μ l;
10×PCR?buffer(-MgCl
2)：1μl；
50mM?MgCl
2：0.3μl；
10mM?dNTP：0.2μl；
20 μ M primer mixtures: 0.2 μ l;
5U/ μ l Taq enzyme: 0.05 μ l;
Aseptic double-distilled water: 5.25 μ l;
Total：10μl。
The pcr amplification condition is:
94 ℃ 3 minutes; 94 ℃ of 30 second, 63 ℃ of 45 second, 72 ℃ of 30 second, 35 circulations; 72 ℃ 7 minutes.Obtain size behind the pcr amplification and be the band of 240bp, the results are shown in accompanying drawing 1.
3.PCR product purification
1) capable 1.5% agarose gel electrophoresis of .PCR product, ultraviolet lamp are observed the band position down, cut glue and weigh.
2). adopt the German MN Nucleospin Extract II of company glue purification test kit purified pcr product, finally use 40 μ l eluant wash-outs.
4.AT clone
Adopt the pGEM-Teasy kit test kit of Promega company to carry out AT clone, operation to specifications.
The configuration linked system:
Teasy?vector：1μl；
2 * connection damping fluid: 5 μ l;
T4 dna ligase: 1 μ l;
PCR product behind the purifying: 1 μ l;
Aseptic double-distilled water: 2 μ l.
4 ℃ of connections are spent the night.
5. connect the conversion of product
1). get 5 μ l and connect product, add 100 μ l DH5a competence bacteriums, mixing left standstill 20 minutes on ice gently.
2). mixture 42 ℃ of circulator bath heat-shockeds 90 seconds, was placed 2 minutes on ice.
3). add the LB nutrient solution of 37 ℃ of pre-temperature of 1ml in the mixture, 37 ℃ of vibrations (180rpm) were cultivated 1 hour.
4). 200 μ l converted products are applied on the LB culture plate (containing 1mol/l IPTG 4 μ l, 20mg/ml X-gal 40 μ l) that contains the 100mg/ml penbritin, are inverted for 37 ℃ and cultivate 10h.
5). picking white colony from the LB culture plate is inoculated in 2ml and contains in the LB nutrient solution of 100mg/ml penbritin, 37 ℃ of vibrations (250rpm) overnight incubation.
6. the evaluation of positive plasmid
1). the enzyme of positive plasmid is cut evaluation
Get 1ml bacterium liquid, the little upgrading grain of alkaline lysis adds the dissolving of 20 μ l aseptic double-distilled waters.Carry out enzyme with EcoR I and cut evaluation, the preparation enzyme is cut system: 10 * buffer H:1 μ l;
EcoR?I(12U/μl)：0.3μl；
Plasmid: 1 μ l;
Aseptic double-distilled water: 7.7 μ l.
37 ℃ of water-bath enzymes were cut 2 hours, and enzyme is cut the endonuclease bamhi that can obtain the 200bp size, proved that sequence is correct.
2). the positive plasmid sequencing
The positive colony 3 that is obtained by step 1 screening is delivered promise match genome research center, Beijing and is carried out gene sequencing, and sequencing result shows that positive colony sequence and known TPMT719 wild-type sequence are in full accord, prove that the positive plasmid sequence is correct.
7. positive plasmid large scale culturing and purifying
The positive colony that screening obtains is inoculated in the 1L LB substratum, after 37 ℃ of shaking table shaking culture are spent the night, adopted the alkaline lysis plasmid purification.Low temperature (20 ℃) is frozen.
TPMT genotype detection process with leukaemic's gene is that the present invention is described in further detail for embodiment below.
We use the purinethol methyl transferase gene type that this test kit detects 20 patients, and flow process is as follows:
1. sample process:
Because sample is genomic dna, adopt the DNA concentration of UV spectrophotometer measuring sample, and with the DNA concentration dilution to 30-100ng/ml
2. dispose A719G amplification mixed solution: PCR reaction mixture: 2.5ul;
A719G primer+probe: 1ul;
H2O：6.5ul；
Amount to: 10ul.
3. the TPMT gene fragment of polymerase chain reaction (PCR) specific amplification:
1) the PCR reaction mixture for preparing is added in the PCR reaction tubes, wherein 719 specific mixtures add reaction tubes.
2) TPMT gene fragment amplification
According to following condition amplification TPMT gene fragment
95 ℃ of pre-sex change 10 minutes; 95 ℃ of sex change 15 seconds, 58 ℃ of annealing 15 seconds, 60 ℃ were extended 45 circulations 45 seconds.
4.PCR the fluorescent scanning of reaction result:
PCR reaction tubes after the amplification is put into ABI 7900HT quantitative fluorescent PCR, use SDS software scans PCR reaction result.Scanning result is seen accompanying drawing 2.
5. the genotypic judgement of sample
Carry out the standard that the sample genotype is judged according to the fluorescent scanning result: the genotype of judging the TPMTA719G pleomorphism site according to the fluorescent scanning result respectively.According to the genotype result of determination, the TPMT enzymic activity.According to the standard that above genotype is judged, can determine the genotype of 20 samples, each sample genotype sees Table 1:
6. carry out curative effect of medication and side effect prediction according to the sample genotype.
According to above-mentioned detected result, the 1st, 5,11,16, No. 19 sample is homozygous mutation, will cause the disappearance of purinethol methyltransgerase enzymic activity.This enzymic activity disappearance will cause the purine medicaments metabolic capacity to reduce significantly, may cause serious toxic reaction as the purine medicaments that uses normal dose, advises that this patient uses the medicine of other type instead.3,6,8,10,14, No. 20 patients are heterozygous, and purinethol methyltransgerase enzymic activity reduces, and the suggestion patient reduces the purine medicaments consumption.
The genotype of table 1.20 sample to be checked
Sequence table
＜110〉Hua'anfo Medicine Research Center Co., Ltd., Beijing
Shenzhen Aosa Medicine Co., Ltd
＜120〉the genotypic test kit of a kind of detection purinethol methyl transferase gene A719G pleomorphism site, method and purposes
<160>4
<170>PatentIn?version?3.3
<210>1
<211>25
<212>DNA
＜213〉synthetic
<400>1
cctcaaaaac?atgtcagtgt?gattt 25
<210>2
<211>30
<212>DNA
＜213〉synthetic
<400>2
cctgatgtca?ttcttcatag?tattttaaca 30
<210>3
<211>21
<212>DNA
＜213〉synthetic
<400>3
tgtaagtaga?tataactttt?c 21
<210>4
<211>20
<212>DNA
＜213〉synthetic
<400>4
taagtagaca?taacttttca 20
Claims (12)
1. the test kit of a fluorescent quantitative poly chain reaction method detection purinethol methyl transferase gene polymorphism is characterized in that, comprises and detects the genotypic Auele Specific Primer of purinethol methyl transferase gene A719G pleomorphism site, probe.
2. test kit as claimed in claim 1 is characterized in that, detection purinethol methyltransgerase A719G pleomorphism site
Forward primer is: 5 '-CCTCAAAAACATGTCAGTGTGATTT-3 ';
Reverse primer is: 5 '-CCTGATGTCATTCTTCATAGTATTTTAACA-3 '.
3. test kit as claimed in claim 1 is characterized in that, detection purinethol methyltransgerase A719G pleomorphism site
Wild-type probe is: VIC-TGTAAGTAGATATAACTTTTC-MGB;
The mutant probe is: FAM-TAAGTAGACATAACTTTTCA-MGB.
4. test kit as claimed in claim 1 is characterized in that, contains PCR reaction mixture and Taq archaeal dna polymerase.
5. test kit as claimed in claim 4 is characterized in that, described PCR reaction mixture contains 100mM Tris-HCl, 100mM KCl, 5.0uM MgCl
2, 0.4mM dATP, 0.4mM dCTP, 0.4mM dGTP, 0.4mM dTTP.
6. test kit as claimed in claim 4 is characterized in that, the content of described Taq archaeal dna polymerase is 1-10U/ul, preferred 5U/ul.
7. test kit as claimed in claim 1 is characterized in that, contains the positive control of purinethol methyltransgerase A719G pleomorphism site wild-type and mutant.
8. test kit as claimed in claim 7 is characterized in that, described positive control is selected from genomic dna, RNA or is cloned into dna fragmentation on the carrier.
9. application rights requires the described test kit of 1-8 to detect the genotypic method of purinethol methyl transferase gene A719G pleomorphism site.
10. method as claimed in claim 9 is characterized in that, the gene amplification program of described detection purinethol methyltransgerase A719G pleomorphism site is:
95 ℃ of pre-sex change 10 minutes; 95 ℃ of sex change 15 seconds, 58 ℃ of annealing 15 seconds, 60 ℃ were extended 45 circulations 45 seconds.
11. as the described test kit of claim 1-8 in prediction curative effect of purine medicaments or the purposes in the side effect.
12. purposes as claimed in claim 11 is characterized in that described purine medicaments is Ismipur, 6-thioguanine or mercapto azoles purine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131776A (en) * | 2013-02-05 | 2013-06-05 | 武汉艾迪康医学检验所有限公司 | A719G single nucleotide polymorphism detection kit of thiopurine methyltransferase (TPMT) * 3C |
| CN104011225A (en) * | 2011-10-21 | 2014-08-27 | 陶氏益农公司 | Method To Determine Zygosity Of The Fad3 Gene In Canola |
| CN104011226A (en) * | 2011-10-21 | 2014-08-27 | 陶氏益农公司 | Method to determine zygosity of the fad2 gene in canola using end-point pcr |
| CN113462758A (en) * | 2021-06-17 | 2021-10-01 | 湖南菲思特精准医疗科技有限公司 | Detection kit for cisplatin metabolic marker, detection method and application thereof |
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2008
- 2008-05-27 CN CNA2008101130878A patent/CN101591697A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104011225A (en) * | 2011-10-21 | 2014-08-27 | 陶氏益农公司 | Method To Determine Zygosity Of The Fad3 Gene In Canola |
| CN104011226A (en) * | 2011-10-21 | 2014-08-27 | 陶氏益农公司 | Method to determine zygosity of the fad2 gene in canola using end-point pcr |
| US9663832B2 (en) | 2011-10-21 | 2017-05-30 | Dow Agrosciences Llc | Method to determine zygosity of the FAD3 gene in canola using end-point taqman® PCR |
| CN103131776A (en) * | 2013-02-05 | 2013-06-05 | 武汉艾迪康医学检验所有限公司 | A719G single nucleotide polymorphism detection kit of thiopurine methyltransferase (TPMT) * 3C |
| CN113462758A (en) * | 2021-06-17 | 2021-10-01 | 湖南菲思特精准医疗科技有限公司 | Detection kit for cisplatin metabolic marker, detection method and application thereof |
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Application publication date: 20091202 |