CN103773859A - SNP marker of mitochondria DNA related to asthenospermia with unknown clinical causes and application thereof - Google Patents
SNP marker of mitochondria DNA related to asthenospermia with unknown clinical causes and application thereof Download PDFInfo
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Abstract
The invention belongs to the fields of genetic engineering and reproductive medicine and discloses an SNP marker of mitochondria DNA related to asthenospermia with unknown clinical causes and application thereof. The marker is a combination of C5601T, T12338C, Al2361G, G13928C, Al5851G, C16179T and G16291A, can be used for preparing an auxiliary diagnosis kit for asthenospermia with unknown clinical causes and has high specificity and sensitivity.
Description
Invention field
The invention belongs to genetically engineered and reproductive medicine field, relate to SNP mark and the application thereof of the Mitochondrial DNA that a kind of and clinical agnogenic azoospermia is relevant.
Background technology
The whole world approximately has the couple at child-bearing age of 8-15% to have growing barrier in various degree, and wherein bridegroom's or husband's side factor accounts for 50%.Research shows, since over half a century, human seminal fluid's quality significantly declines, and what think at present male sterility is multifactor, a multistage process, is environmental risk factors (external cause) and the coefficient result of idiogenetics factor (internal cause).Wherein, idiogenetics factor comprises that the chromosomal inheritance factor changes, the abnormal and Mitochondrial Genome Overview genetic construction variation of epigenetic modification.Mitochondrial Genome Overview is as the outer unique genome of core, and its genetic construction variation is considered to one of most important inherited pathogenic factor of male sterility.
Plastosome (mitochondria) is a complicated organoid, is the main source of cells play function institute energy requirement.Mitochondrial DNA (mitochondrial DNA, mtDNA) be a double-stranded closed hoop molecule, 16569 base pairs (accession number is NC_012920.1) are comprised, 37 negative gene responsible editor codes 13 respiratory chain enzyme complex subunits, 22 kinds of transfer RNAs (tRNA) and 2 kinds of ribosome-RNA(rRNA)s (rRNA) on it.Owing to lacking protection and the DNA repair system of histone, the mutation rate of mtDNA is 10-100 times of core DNA.ATP is the primary energy derive of motility of sperm, and it is mainly from two approach: anaerobic glycolysis and mitochondrial respiratory chain OXPHOS(aerobic oxidation) approach.Classical theory thinks that the mitochondrial sheath that sperm stage casing plastosome forms supplies with institute's energy requirement in sperm motility process, allly affects any factor that ATP generates, as respiratory enzyme inhibitor etc. all can directly or indirectly affect motility of sperm.Studies have shown that, the sperm before capacitation mainly relies on anaerobic glycolysis energy supply, and this process does not need plastosome to participate in.After capacitation, Metabolic activity obviously strengthens, and shows that in mitochondria of sperms, OXPHOS produces a large amount of ATP, and one species specific " whipping sample " mode of motion appears in sperm, and by this kind of mode of motion, sperm is fertilized through ovum zona pellucida.As can be seen here, in the time that the attack of the radical pair mtDNA such as active oxygen (ROS) causes oxidative damage or mtDNA sudden change to affect the generation of ATP, will affect motility of sperm, cause growing barrier.
At present, the tentative diagnosis method of azoospermia (asthenospermia) is mainly disease history inquire, physical examination, seminal parameters, seminal plasma biochemistry, blood hormone detection, B ultrasonic and karyomit(e) detection etc.The World Health Organization is at the deagnostic test of male sterility and process handbook and indicate: azoospermia refer to seminal parameters in the sperm (a and b level) of propulsion be less than 50% or the sperm of a level motion illness that is less than 25%, azoospermia claims again low sperm activity.(artificial counting or utilize computer aided system analysis is differentiated in the conventional motility of sperm analysis at present main motility of sperm that relies on, be CASA), there is no other effective means, but himself there is certain defect, as larger in individual semen quality fluctuation, especially be vulnerable to the impact of the factors such as ascetic number of days, temperature, collecting semen method, thereby it is inaccurate to cause motility of sperm to measure; Early diagnosis effect to corresponding disease also far can not be satisfied the demand.Although investigator is just making great efforts to explore new azoospermia diagnostic biomarkers both at home and abroad at present, and existing evaluation measures is effectively supplemented, but be difficult to break through the bottleneck in the development of traditional biological mark and male genetic practical application, i.e. Analysis of Semen Quality result fluctuation, early diagnosis difficulty etc. always.In addition, due to etiology unknown, lack effective treatment means, most of azoospermia patients can only select the assisted reproduction such as ICSI or AID means, not only cannot meet psychological requirement, and likely occur offspring's hereditary defect.
Research shows, inherited genetic factors is the principal element that causes male sterility, and mitochondrial inheritance variation plays an important role therein.It is modal one in the heritable variation of the mankind.The existence of heritable variation has been considered to give individual different phenotypic character, and for the differential responses of the factor such as environmental exposure, pharmacological agent, therefore heritable variation may be to cause the individual important hereditary basis to common disease morbidity and prognosis susceptibility difference.Also inheritance of mitochondrion DNA variation is not applied at present to the report of azoospermia diagnosis, if can filter out the inheritance of mitochondrion DNA of azoospermia susceptible makes a variation as biomarker, and develop corresponding diagnostic kit, will be once strong promotion to the diagnosis present situation of azoospermia, also for its drug screening, evaluating drug effect and targeted therapy have been opened up new approach.
Summary of the invention
The object of the invention is for above-mentioned technical problem, propose the inheritance of mitochondrion DNA variable nidicant thing that a kind of and clinical agnogenic azoospermia auxiliary diagnosis is relevant and the gene that contains this mark.
Second object of the present invention is to provide the specificity amplification primer of above-mentioned inheritance of mitochondrion DNA variable nidicant thing.
The 3rd object of the present invention is to provide the specificity of above-mentioned inheritance of mitochondrion DNA variable nidicant thing and extends primer.
The 4th object of the present invention is to provide above-mentioned inheritance of mitochondrion DNA variable nidicant thing and specificity amplification primer and specificity thereof and extends primer in the application of preparing in azoospermia auxiliary diagnostic box.
The 5th object of the present invention is to provide azoospermia auxiliary diagnostic box.
Contriver is by separating and study clinical agnogenic Asthenospermia and contrasting peripheral blood inheritance of mitochondrion DNA variation (SNP) with the healthy male of its age-matched, finding one group makes a variation with the high specific of azoospermia height correlation and the inheritance of mitochondrion DNA of susceptibility, and develop the azoospermia auxiliary diagnostic box that can be convenient to clinical application, for examination and the diagnosis of clinical agnogenic azoospermia provide Data support, for finding to have the new small molecule drug provision Data support of potential therapeutic value.
The object of the invention is to realize by following technical proposal:
A SNP mark for the Mitochondrial DNA relevant to clinical agnogenic azoospermia auxiliary diagnosis, the C5601T that this SNP mark is Mitochondrial DNA, T12338C, A12361G, G13928C, A15851G, C16179T, G16291A.
A gene relevant to clinical agnogenic azoospermia auxiliary diagnosis, the sequence of this gene is that accession number is to have following SNP:C5601T, T12338C in the mtdna sequence of NC_012920.1, A12361G, G13928C, A15851G, C16179T, G16291A.
For detection of the specificity amplification primer of described inheritance of mitochondrion DNA variable nidicant thing, these primers are:
The primer sequence of C5601T is SEQ ID No:1 and SEQ ID No:2; The primer sequence of T12338C is SEQ ID No:4 and SEQ ID No:5; The primer sequence of T12361G is SEQ ID No:7 and SEQ ID No:8; The primer sequence of G13928C is SEQ ID No:10 and SEQ ID No:11; The primer sequence of A15851G is SEQ ID No:13 and SEQ ID No:14; The primer sequence of C16179T is SEQ ID No:16 and SEQ ID No:17; The primer sequence of G16291A is SEQ ID No:19 and SEQ ID No:20.
Specificity for detection of described inheritance of mitochondrion DNA variable nidicant thing is extended primer sequence, and these extension primer sequences are:
The extension primer sequence of C5601T is SEQ ID No:3; The extension primer sequence of T12338C is SEQ ID No:6; The extension primer sequence of A12361G is SEQ ID No:9; The extension primer sequence of G13928C is SEQ ID No:12; The extension primer sequence of A15851G is SEQ ID No:15; The extension primer sequence of C16179T is SEQ ID No:18; The extension primer sequence of G16291A is SEQ ID No:21.
The application of described SNP mark in the clinical agnogenic azoospermia auxiliary diagnostic box of preparation.
The application of described gene in the clinical agnogenic azoospermia auxiliary diagnostic box of preparation.
Described specificity amplification primer and/or specificity are extended the application of primer in the clinical agnogenic azoospermia auxiliary diagnostic box of preparation.
A kind of clinical agnogenic azoospermia auxiliary diagnostic box, this test kit is for detection of C5601T in peripheral blood DNA, T12338C, A12361G, G13928C, A15851G, C16179T, G16291A.
Described diagnostic kit, this test kit contains described specificity amplification primer and/or described specificity is extended primer.
Described diagnostic kit, this test kit also comprises the reagent that round pcr is conventional.
Specifically, the technical scheme that the present invention deals with problems comprises: (1) sets up sample storehouse and the database of unified standard: gather standard compliant blood sample with Standard operation procedure SOP (SOP), demography data and clinical data that systematic collection is complete.(2) genotype detection: select clinical agnogenic azoospermia case, contrast with the healthy male of clinical agnogenic azoospermia case age-matched, utilize Illumina sequencing technologies, in the full genome range of plastosome, find out the inheritance of mitochondrion DNA variation relevant to clinical agnogenic azoospermia.(3) to the positive association line plastochondria heritable variation filtering out, further in large sample crowd, detect, with the stability that judges that it is associated.(4) development of azoospermia auxiliary diagnostic box: according to the genotype distribution frequency mitochondrial inheritance that there were significant differences variation exploitation mitochondrial inheritance variation auxiliary diagnostic box in azoospermia case and healthy male contrast.
The inventor gathers standard compliant blood sample with Standard operation procedure SOP (SOP), demography data, clinical data etc. that systematic collection is complete, and having adopted Illumina sequencing technologies to carry out the full genome scanning of plastosome, SNaPshot gene type carries out the detection of Single locus etc.
The experimental technique of research mainly comprises following components specifically:
1. the selection of research sample
(1) repeat semen quality and detect, be diagnosed as azoospermia;
(2) sexual function is normal; Get rid of have that cryptorchidism medical history, testitis, vas deferens are blocked, the abnormal and Y chromosome Azoospermia factor of vasotomy, polysomy is micro-deleted etc. has the patient of the known cause of disease
(3) contrast with the healthy male of case age-matched
This research adopts 1724 routine standard compliant samples to study altogether.
2. phenol-chloroform method extracts peripheral blood genomic dna, according to a conventional method operation.Conventionally can obtain 20-50ng/ μ l DNA, purity (ultraviolet 260OD and 280OD ratio) is at 1.6-2.0.
3. Mitochondrial DNA genome checks order entirely
(1) get experimenter's complete genome DNA sample;
(2) utilize Illumina sequencing technologies to carry out the full genome scanning of plastosome;
(3) detection the difference difference of more each genotype in azoospermia case contrasts with healthy male.
4. the SNaPshot gene type in heritable variation site
(1) get experimenter's DNA sample;
(2) design specificity multiple PCR primer and the single-basic extension primer of single heritable variation;
(3) carry out multi-PRC reaction and extension;
(4) detect and more clinical agnogenic azoospermia case contrast with healthy male in the distributional difference of different genotype.
5. diagnostic reagent box preparation method
Utilize Illumina sequencing technologies carry out determining after full genome scanning and single heritable variation detect clinical agnogenic azoospermia case contrast with healthy male in the heritable variation that there were significant differences of genotype distribution frequency, the index of diagnosing as clinical agnogenic azoospermia.The heritable variation of falling ill relevant with the clinical agnogenic azoospermia composition auxiliary diagnostic box (for detection of C5601T, T12338C, A12361G, G13928C, A15851G, C16179T, G16291A) finally filtering out.Diagnostic reagent can comprise that the Auele Specific Primer of these variant sites and specificity extend primer, and the reagent such as Taq enzyme, dNTP.
6. statistical analysis technique
The difference of using chi square test (for classified variable) or student t check (for continuous variable) comparison demographic characteristics etc. to distribute between research object group.Carry out association analysis with the additive model in Logistic regression analysis.
In order further to study the comprehensive indication of these 7 heritable variations formations for the effect of early diagnosis, we have built a mathematical formula, consider positive and negative associated situation and the relation intensity fallen ill with clinical agnogenic azoospermia in each heritable variation site.Specifically, we mark to two kinds of genotype in each heritable variation site, wild homozygous=" 0 ", homozygous=" 2 " make a variation, regression coefficient under additive model during take single snp analysis is weight, and the situation that considers each SNP is determined a dangerous score value to each research object.The method of calculation of dangerous score value are as follows: dangerous score value=(scoring of 0.95 × C5601T)+(scoring of 1.18 × T12338C)+(scoring of 1.00 × A12361G)+(scoring of 0.48 × G13928C)+(scoring of 1.30 × A15851G)+(scoring of 1.10 × C16179T)+(scoring of 0.90 × G16291A), the danger of acquisition divides in the 470 routine samples that value coefficient and boundary value be applied directly to genome-wide association study.(take C5601T as example: the regression coefficient (in table 1) of 0.95 as C5601T variant sites; The scoring of C5601T, because plastosome only has 2 kinds of genotype, if this point for CC(wild homozygous) assignment be 0 minute, if TT(variation is homozygous) assignment be 2 points.Certain SNP is wild homozygous or make a variation and homozygously determined by instrument detected result; The overall score of certain sample is these 7 summations that SNP marks respectively, and the genotype of single SNP is just calculated a pilot process of scoring, does not need to know concrete genotype.）
Statistical analysis all completes (PLINK1.07) by special statistical analysis software.The horizontal P value of significance,statistical is made as 0.05, and all statistical test are two-tailed test.
Below further instruction of the present invention:
In the 236 qualified clinical agnogenic azoospermia cases of example and 234 routine healthy male contrasts, two groups of age equilibriums are comparable.We carry out the full genome scanning of plastosome by these two groups of crowds through Illumina sequencing technologies and obtain correlated results.
According to full genome detected result, the inventor detects that the heritable variation that genotype distribution frequency there are differences in " clinical agnogenic azoospermia case " group and " healthy male contrast " group comprises: C5601T, T12338C, A12361G, G13928C, A15851G, C16179T, G16291A.
According to above-mentioned detected result, we fall ill relevant heritable variation in the other 688 clinical agnogenic azoospermia cases of example by these 7 to clinical agnogenic azoospermia and in contrasting with 566 routine healthy males of its age-matched, have carried out the detection of single heritable variation, and result detects consistent with full genome.
Single factor and logistic Regression Analysis result all show, these 7 heritable variations exist remarkable associated with the morbidity of clinical agnogenic azoospermia, 7 heritable variations are all Hazard Factor, and variation allelotrope can increase the onset risk of clinical agnogenic azoospermia.
Further analyze the combination of these 7 heritable variations for the effect of clinical agnogenic azoospermia diagnosis, find that its combination can be good at distinguishing case and contrasts.
According to above-mentioned experimental result, the inventor has prepared a kind of test kit that can be used for clinical agnogenic azoospermia auxiliary diagnosis, comprises Auele Specific Primer, specificity extension primer and/or other detection reagent of measuring above-mentioned heritable variation in experimenter's blood specimen DNA.
Particularly, the combination of these 7 heritable variations, or the dependent diagnostic test kit constituting of the Auele Specific Primer of these 7 heritable variations and specificity extension primer contributes to the auxiliary diagnosis of clinical agnogenic azoospermia, for clinician quick and precisely grasps patient's morbid state and coincident with severity degree of condition, take in time the scheme of preventing and treating of more personalized to provide support.
Beneficial effect of the present invention:
Heritable variation mark provided by the invention is as the superiority of the mark of clinical agnogenic azoospermia auxiliary judgment:
(1) heritable variation site is a kind of novel gene biomarker, be different from traditional biological mark, stable, Wicresoft, be easy to detect, susceptibility and the specificity of medical diagnosis on disease will be improved greatly, brand-new situation is started in diagnosis and treatment for clinical agnogenic azoospermia by the successful exploitation of such biomarker, for the development of other diseases biomarker is offered reference.
(2) heritable variation site kit is a kind of system, comprehensive diagnostic kit, can be used for the auxiliary diagnosis of clinical agnogenic azoospermia, for clinician quick and precisely grasps conditions of patients, takes the scheme of preventing and treating of more personalized to provide support in time.
(3) adopt tight checking and appraisement system, inventor's initial stage adopts genome sequencing to obtain the heritable variation spectrum of disease-related, and applies SNaPshot methods of genotyping and verify in large sample; Above method and tactful application acceleration and guaranteed heritable variation biomarker and diagnostic kit application are clinically also the reference on development supplying method and the strategy of other diseases biomarker.
The present invention is by controlling the influence factor to disease progression such as age, research heritable variation is in the application prospect of clinical agnogenic azoospermia auxiliary diagnosis, set forth the impact of heritable variation for clinical agnogenic azoospermia progress, disclose its diagnostic value.Therefore, the present invention has obtained clinical agnogenic azoospermia morbidity correlative heritability spectrum and Specific marker; By the development and application of heritable variation biomarker and diagnostic kit, can make the diagnosis of clinical agnogenic azoospermia more convenient and easy, for clinician quick and precisely grasps conditions of patients, for clinical therapeutic efficacy evaluation lays the foundation, and for finding that the new small molecule drug targets with potential therapeutic value offers help.
Accompanying drawing explanation
Fig. 1 is the ROC curve between healthy male control group and clinical agnogenic azoospermia case group.
Embodiment
The collection of embodiment 1 sample and the arrangement of sample data
Contriver starts to have collected in January, 2011 from Reproductive Medicine Center of Nanjing Medical University a large amount of clinical agnogenic Asthenospermia blood specimens 4 months June in 2007, by the arrangement to sample data, contriver has therefrom selected 1724 examples to meet the full genome chip scanning of sample of following standard and the laboratory sample of single SNP SNaPshot gene type:
1, repeat semen quality and detect, be diagnosed as azoospermia;
2, sexual function is normal; Get rid of have that cryptorchidism medical history, testitis, vas deferens are blocked, the abnormal and Y chromosome Azoospermia factor of vasotomy, polysomy is micro-deleted etc. has the patient of the known cause of disease;
3, contrast with the healthy male of case age-matched.
And system acquisition the situation such as demography data and clinical data of these samples.
The full genome scanning of SNP in embodiment 2 peripheral blood DNAs
In the above-mentioned qualified 236 clinical agnogenic Asthenospermias of example and 234 routine healthy male contrasts, two groups of age-matched.By these two groups of crowds through the Illumina acquisition correlated results that entirely checks order.Concrete steps are:
1, to being stored in hemocyte in 2ml cryopreservation tube, to add haemolysis reagent (be lysate, 40 deal collocation methods are as follows: after sucrose 219.72g, magnesium chloride 2.02g and triton x-100 (amresco0694) 20ml mix, be settled to 2000ml with TrisHcl solution, lower with), put upside down to mix and proceed to completely afterwards.
2, remove red corpuscle: with haemolysis reagent, 5ml centrifuge tube is mended to 4ml, put upside down and mix, centrifugal 10 minutes of 4000rpm, abandons supernatant.In precipitation, add 4ml haemolysis reagent, again put upside down and mix cleaning once, centrifugal 10 minutes of 4000rpm, abandons supernatant.
3, extracting DNA: add 1ml extract and (contain 122.5ml 0.2M sodium-chlor in every 300ml in precipitation, 14.4ml 0.5M ethylenediamine tetraacetic acid (EDTA), 15ml 10% sodium lauryl sulphate, 148.1ml distilled water, lower same) and 8 μ l Proteinase Ks, on oscillator, fully concussion mixes, and 37 ℃ of water-baths are spent the night.
4, remove protein: add 1ml Tris-balance phenol and fully mix (have gentle hands is shaken 15 minutes), centrifugal 10 minutes of 4000rpm, gets supernatant and proceed in new 5ml centrifuge tube.In supernatant liquor, add equal-volume chloroform and primary isoamyl alcohol mixed solution (chloroform: primary isoamyl alcohol=24:1, v/v, lower with), fully mix after (hand 15 minutes), centrifugal 10 minutes of 4000rpm, gets supernatant (being divided into the centrifuge tube of two 1.5ml).
5, DNA precipitation: in supernatant liquor, add the sodium-acetate 60 μ l of 3M, then add and the isopyknic ice dehydrated alcohol of supernatant liquor, upper and lower jog, visible white flocculent precipitate, then with the centrifugal 10min of 12000rpm.
6, DNA washing: add ice dehydrated alcohol 1ml in precipitation, the centrifugal 10min of 12000rpm, abandons supernatant final vacuum and drain or be placed in clean dry environment evaporate to dryness.
7, measure concentration: conventionally can obtain 20-50ng/ μ l DNA, purity (ultraviolet 260OD and 280OD ratio) is at 1.6-2.0.
8, entirely check order: utilize Illumina sequencing technologies to carry out the full genome scanning of plastosome.
9, data analysis and processing: the genotype distribution frequency heritable variation that there were significant differences of finding in " clinical agnogenic azoospermia case " group and " healthy male contrasts " group is enumerated out hereinbefore, the results are shown in Table 1.
Table 1: case group and control group whole-genome association result
The SNaPshot gene type of embodiment 3 single heritable variations
Above-mentioned full genome scanning is found to detect in the other 688 clinical agnogenic azoospermia cases of example and 566 routine healthy males contrast with the clinical agnogenic azoospermia relevant SNP that falls ill, and concrete steps are:
1, add haemolysis reagent to the hemocyte being stored in 2ml cryopreservation tube, put upside down to mix and proceed to completely afterwards.
2, remove red corpuscle: with haemolysis reagent, 5ml centrifuge tube is mended to 4ml, put upside down and mix, centrifugal 10 minutes of 4000rpm, abandons supernatant.In precipitation, add 4ml haemolysis reagent, again put upside down and mix cleaning once, centrifugal 10 minutes of 4000rpm, abandons supernatant.
3, extracting DNA: add 1ml extract and 8 μ l Proteinase Ks in precipitation, on oscillator, fully concussion mixes, and 37 ℃ of water-baths are spent the night.
4, remove protein: add 1ml Tris-balance phenol and fully mix (have gentle hands is shaken 15 minutes), centrifugal 10 minutes of 4000rpm, gets supernatant and proceed in new 5ml centrifuge tube.In supernatant liquor, add equal-volume chloroform and primary isoamyl alcohol mixed solution (chloroform: primary isoamyl alcohol=24:1), after fully mixing (hand 15 minutes), centrifugal 10 minutes of 4000rpm, gets supernatant (being divided into the centrifuge tube of two 1.5ml).
5, DNA precipitation: in supernatant liquor, add the sodium-acetate 60 μ l of 3M, then add and the isopyknic ice dehydrated alcohol of supernatant liquor, upper and lower jog, visible white flocculent precipitate, then with the centrifugal 10min of 12000rpm.
6, DNA washing: add ice dehydrated alcohol 1ml in precipitation, the centrifugal 10min of 12000rpm, abandons supernatant final vacuum and drain or be placed in clean dry environment evaporate to dryness.
7, measure concentration: conventionally can obtain 20-50ng/ μ l DNA, purity (ultraviolet 260OD and 280OD ratio) is at 1.6-2.0.
8, carry out SNaPshot gene type
(1) multi-PRC reaction and purifying
Multiplex PCR system: DNA profiling 1 μ l; Faststar Taq (Roche) archaeal dna polymerase 0.1 μ l; 10XFaststarTaq Buffer (Roche) 1 μ l; DNTP mixture 1.5 μ l; MgCl
21 μ l; (every couple of primer 0.2 μ l), uses ddH to multiple PCR primer mixed solution 1 μ l
2o supplies 10 μ l.Multiplex PCR cycling condition program: 95 ℃ * 4min-[94 ℃ * 30sec-60 ℃ (0.5 ℃/cycle) * 50sec-72 ℃ of * 90sec] * 11cycles-[94 ℃ * 30sec-55 ℃ * 50sec-72 ℃ * 90sec] 4 ℃ of * ∞ of 72 ℃ of * 10min – of * 11cycles –.(amplimer sequence is in table 2)
Purifying after subsequently PCR product being detected by 2% agarose gel electrophoresis.Purification process is: Phosphatase alkaline shrimp (SAP, Roche) 1 μ l, and ExonukleaseI (ExoI, Amershampharmacia) 0.4 μ l, amplified production 1 μ l, uses ddH
2o supplies 4 μ l, mixes rear 37 ℃ of 60min, and 85 ℃ of 15min finish rear 4 ℃ of preservations.
(2) single base extension and purifying
(6 μ are l) as follows: extend primer mixture 0.5 μ l (every primer final concentration is 0.1 μ M) for single base extension system; Multiplex PCR purified product 1-2 μ l; SNaPshot Mutiplex (ABI) 0.7-1 μ l; 5X Sequencing Buffer (BigDye) 1 μ l, ddH
2o supplies 6 μ l.Extension program: 96 ℃ * 1min-[96 ℃ * 10sec-52 ℃ * 50sec-60 ℃ of * 30sec] * 28cycles-4 ℃ * ∞.(extending primer sequence in table 3)
In extension products, add subsequently 1 μ l SAP to carry out purifying, purifying procedure: 37 ℃ of 60min, 85 ℃ of 15min, finish rear 4 ℃ of preservations.
9, genotype interpretation: the extension product of getting 1 μ l purifying, 0.2 μ l GeneScan-120Liz Size Standard (ABI), add DNA denaturing agent methane amide (Hi-Di Formamide) cumulative volume 10 μ l/ holes, mix rear 95 ℃ of sex change 5 minutes, put immediately cooled on ice.Denatured products is splined on ABI 3130XL sequenator and carries out gene type subsequently, operation Genescan V3.7 analyzes experimental result, interpretation record data.
10, data processing and analysis: utilize the difference of two kinds of genotype distribution frequency in case group and control group of the more each heritable variation of addtive model in Logistic regression model, result is basically identical with full genome scanning data results.
The PCR primer information of table 2 related SNP
Table 3 related SNP is extended primer information
Embodiment 4 utilizes risk assessment separating method further to analyze SNP and clinical agnogenic azoospermia morbidity
According to the above results, the inventor is by the comparison to 2 groups of samples (" clinical agnogenic azoospermia case group " and " healthy male control group ") genotype distribution frequency, select positive associated SNP, take single SNP regression coefficient in full genome scanning sample as weight, further try to achieve dangerous score value, draw ROC and assess susceptibility and the specificity of prediction, and then assess the judgement of these SNP to clinical agnogenic azoospermia morbidity.The Conjoint Analysis of 7 SNP marks is found, these 7 SNP separate healthy male control group and clinical agnogenic azoospermia case group with 71.61% AUC, and the sensitivity of best stagnation point is 77.97%, specific degree: 61.11%(Fig. 1).
Therefore, the inventor has proved employing C5601T, T12338C, and A12361G, G13928C, A15851G, C16179T, the combination of G16291A can be distinguished healthy male contrast and clinical agnogenic Asthenospermia well.
Embodiment 5 is for the making of clinical agnogenic azoospermia auxiliary diagnosis SNP test kit
The making of SNP test kit and operating process are based on the full order-checking of Illumina genome and SNaPshot genotyping technique.Test kit contains a collection of heritable variation specificity amplification primer, and (primer sequence that comprises following primer: C5601T is SEQ ID No:1 and SEQ ID No:2; The primer sequence of T12338C is SEQ ID No:4 and SEQ ID No:5; The primer sequence of T12361G is SEQ ID No:7 and SEQ ID No:8; The primer sequence of G13928C is SEQ ID No:10 and SEQ ID No:11; The primer sequence of A15851G is SEQ ID No:13 and SEQ ID No:14; The primer sequence of C16179T is SEQ ID No:16 and SEQ ID No:17; The primer sequence of G16291A is SEQ ID No:19 and SEQ ID No:20).Specificity is extended primer sequence, and (the extension primer sequence that comprises following extension primer sequence: C5601T is SEQ ID No:3; The extension primer sequence of T12338C is SEQ ID No:6; The extension primer sequence of A12361G is SEQ ID No:9; The extension primer sequence of G13928C is SEQ ID No:12; The extension primer sequence of A15851G is SEQ ID No:15; The extension primer sequence of C16179T is SEQ ID No:18; The extension primer sequence of G16291A is SEQ ID No:21), can also there is the required common agents of corresponding round pcr, as: dNTPs, MgCl
2, distilled water, Taq enzyme etc., these common agents are all well known to those skilled in the art, can also have in addition standard substance and contrast (as determined genotypic standard substance and blank etc.).The value of this test kit is only to need peripheral blood and does not need other tissue sample, by simplifying most with special primer and extending primer and detect heritable variation, compose the clinical agnogenic azoospermia of auxiliary judgment by heritable variation again, not only stable, easy to detect, and accurately, greatly improve susceptibility and the specificity of medical diagnosis on disease, therefore this test kit is dropped into practice, can help to instruct diagnosis and more effective individualized treatment.
Claims (10)
1. a SNP mark for the Mitochondrial DNA relevant to clinical agnogenic azoospermia auxiliary diagnosis, is characterized in that this mark is C5601T, T12338C, A12361G, G13928C, A15851G, C16179T, the combination of G16291A.
2. a gene relevant to clinical agnogenic azoospermia auxiliary diagnosis, the sequence that it is characterized in that this gene is that accession number is to have following SNP:C5601T, T12338C in the mtdna sequence of NC_012920.1, A12361G, G13928C, A15851G, C16179T, G16291A.
3. for detection of the specificity amplification primer of SNP mark claimed in claim 1, it is characterized in that this amplimer is:
The primer sequence of C5601T is SEQ ID No:1 and SEQ ID No:2; The primer sequence of T12338C is SEQ ID No:4 and SEQ ID No:5; The primer sequence of T12361G is SEQ ID No:7 and SEQ ID No:8; The primer sequence of G13928C is SEQ ID No:10 and SEQ ID No:11; The primer sequence of A15851G is SEQ ID No:13 and SEQ ID No:14; The primer sequence of C16179T is SEQ ID No:16 and SEQ ID No:17; The primer sequence of G16291A is SEQ ID No:19 and SEQ ID No:20.
4. extend primer for detection of the specificity of SNP mark claimed in claim 1, it is characterized in that this extension primer is:
The extension primer sequence of C5601T is SEQ ID No:3; The extension primer sequence of T12338C is SEQ ID No:6; The extension primer sequence of A12361G is SEQ ID No:9; The extension primer sequence of G13928C is SEQ ID No:12; The extension primer sequence of A15851G is SEQ ID No:15; The extension primer sequence of C16179T is SEQ ID No:18; The extension primer sequence of G16291A is SEQ ID No:21.
5. the application of SNP mark claimed in claim 1 in the clinical agnogenic azoospermia auxiliary diagnostic box of preparation.
6. the application of gene claimed in claim 2 in the clinical agnogenic azoospermia auxiliary diagnostic box of preparation.
7. specificity amplification primer claimed in claim 3 and/or specificity claimed in claim 4 are extended the application of primer in the clinical agnogenic azoospermia auxiliary diagnostic box of preparation.
8. a clinical agnogenic azoospermia auxiliary diagnostic box, is characterized in that this test kit is for detection of C5601T in peripheral blood DNA, T12338C, A12361G, G13928C, A15851G, C16179T, G16291A.
9. diagnostic kit according to claim 7, is characterized in that this test kit contains specificity amplification primer claimed in claim 2 and/or specificity claimed in claim 3 is extended primer.
10. diagnostic kit according to claim 8 or claim 9, is characterized in that this test kit also comprises the reagent that round pcr is conventional.
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| CN104561279A (en) * | 2014-12-22 | 2015-04-29 | 江苏省家禽科学研究所 | Method of improving quality of chicken semen, primer used for method, kit and using method of kit |
| CN104561279B (en) * | 2014-12-22 | 2017-01-18 | 扬州翔龙禽业发展有限公司 | Method of improving quality of chicken semen, primer used for method, kit and using method of kit |
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| CN106990177A (en) * | 2017-03-29 | 2017-07-28 | 山东大学 | Purposes of the glutamine of 617 generation mass shifts of AKAP4 albumen in the few weak smart diagnostic reagent of severe is prepared |
| CN107024553A (en) * | 2017-03-29 | 2017-08-08 | 山东大学 | Purposes of the serine of 8 generation mass shifts of AKAP3 protein 20s in the few weak smart diagnostic reagent of severe is prepared |
| CN107805637A (en) * | 2017-11-23 | 2018-03-16 | 黄志清 | The RanBP9 mutators and its application that 133 sites are undergone mutation |
| CN108866183A (en) * | 2018-08-25 | 2018-11-23 | 右江民族医学院附属医院 | The relevant GRP78 gene SNP marker of asthénospermie and its application |
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