CN101775435B - Method and kit for detecting polymorphism of mitochondrial ND1 gene mononucleotide, and application of kit - Google Patents
Method and kit for detecting polymorphism of mitochondrial ND1 gene mononucleotide, and application of kit Download PDFInfo
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Abstract
The invention discloses a method for detecting SNP of mitochondrial ND1 gene mt DNA 3970 locus, a kit for detecting the SNP of the locus and application of the kit. The method does not have special restriction on samples, namely, either body fluid or histiocyte can be used as a detection sample, so the detection is convenient and rapid; auele specific primers designed by the method do not have strict requirement on the length of basic group of a region to be amplified, and PCR products can be obtained and high-resolution melting curve can be analyzed by using the auele specific primers; and before PCR reaction, saturated fluorescent dye is added, a light scanner detects fluorescent signals by optics and draws a temperature melting curve, so that wild type, heterozygous mutation and homozygous mutation can be accurately differentiated according to the curve. The invention has the advantages of simple and convenient operation, low detection cost, accurate detection result, and high application and market values.
Description
Technical field
The present invention relates to the method for a kind of detection line plastochondria ND1 gene mononucleotide polymorphism (SNP), the method for the SNP in specifically a kind of detection line plastochondria ND1 gene mtDNA 3970 sites and the application that detects test kit He this test kit of this site SNP.
Background technology
Good health and a long life are the focuses of various countries' research always, and this is because long lived elder is not suffered from old and feeble relevant degenerative disease such as apoplexy in the process of aging, cardiovascular disorder, diabetes B, Parkinson's disease, senile dementia, cancer etc., and healthy life.These old and feeble relevant degenerative diseases not only bring heavy economical load, influence quality of life to the patient; Simultaneously since China since 1998 get into aging society; The aged accounts for total man's mouth constituent ratio (10%) and is increasing year by year, expects the year two thousand twenty, and China will account for 27% of total man's mouth greater than 60 years old population; So also can cause huge pressure, will become the significant medical problem that influences Chinese society welfare and health economy to society.The long-lived Mechanism Study can be given a clue for the pathomechanism of diseases associated with senescence also can predict the onset risk of diseases associated with senescence.
With suffering from old and feeble relevant degenerative disease is a kind of complex inheritance proterties that receives a plurality of h and E factor affecting, comprises the interaction that reaches between the gene between gene and the environment.Gene with (or) environment promotes longevity through reducing old and feeble relevant degenerative disease or delaying senility.But influencing the definite factor of long-lived does not illustrate up to now as yet.Twin study shows that long-lived phenotype 25% is by inherited genetic factors decision (Christensen K; Johnson TE; Vaupel JW.The quest for genetic determinants of human longevity:challengesand insights.Nature, 2006; 7:436-448).Simultaneously; Siblings' the life-span that also shows the centenarian in the pedigree analysis is than high about 4 times of (the Christensen K of general crowd; Johnson TE; Vaupel JW.Thequest for genetic determinants of human longevity:challenges andinsights.Nature, 2006; 7:436-448).These researchs have proved that inherited genetic factors plays an important role in long-lived mechanism.And, in the association study of case-control, found some genetic markers relevant with longevity, as No. 4 karyomit(e) and some genes as, APOE, HLA, mtDNA etc.
Because vital role and the mitochondrial sudden change of plastosome in cell can cause old and feeble relevant disease (Audesh Bhat; Anil Koul; Swarkar Sharma; Et al.The possible role of 10398Aand 16189C mtDNA variants in providing susceptibility to T2DM in two NorthIndian populations:a replicative study.Hum Genet; 2007,120:821-826.), so Mitochondrial DNA possibly play important effect in long-lived mechanism.The more important thing is that old and feeble relevant disease shows as matrilinear inheritance more, the mode of inheritance of this and Mitochondrial DNA is closely similar.Like this, we infer that long-lived crowd has been hereditary mother's Mitochondrial DNA, and being expressed in of these Mitochondrial DNAs can protect the long-lived crowd must not diseases associated with senescence on the function, thereby have prolonged its life-span.Like this, the Mitochondrial DNA variation is more and more receiving investigator's concern with old and feeble relevant degenerative disease relation.Plastosome is the minicell device in the cytoplasm, contains required albumen and synthetic required tRNA and the rRNA of these albumen of double-stranded cyclic DNA coding oxidative phosphorylation process of oneself.Each cell contains thousands of plastosomes, contains 2-10 DNA in each plastosome, therefore, contains tens thousand of DNA copies in each cell.The degenerative disease relevant with aging that carries out at present proves because of studying the association analysis methods that adopt more, is effective with SNPs as genomic marker.Like the SNP5178A and long-lived relevant (Tanaka, M., Gong, J.S. that in the long-lived colony of Japan, finds the Mitochondrial DNA coding region; Zhang, J., Yoneda, M.; AndYagi, K.Mitochondrial genotype associated with longevity.Lancet, 1998; 351,185-186).After this many researchs both domestic and external have confirmed that further this mutational site of 5178A is with long-lived relevant.Other and long-lived relevant SNPs such as 9055A (Ross OA have also been found subsequently; McCormack R, Curran MDet al:Mitochondrial DNA polymorphism:its role in longevity of the Irishpopulation.Exp Gerontol 2001; 36:1161-1178.).
SNP is meant the dna sequence polymorphism that single nucleotide diversity causes on the genomic level, and the frequency in the crowd needs＞1%.Have 70.1% to be the conversion between the homotype base during this single base of SNPs changes: like G/A or T/C, 29.1% for occurring in the transversion between purine and the pyrimidine.C (cytosine(Cyt)) is the most labile site in the human genome, because great majority are the cytosine(Cyt)s that methylate, can convert T (thymus pyrimidine) into by spontaneous deaminizating, and SNPs has comprised the 80-90% of known polymorphum, is modal heritable variation.Because the selective pressure of existence causes the distribution of SNP in term single gene and whole genome to be ununiformity.SNPs is 4 times of coding region in the quantity of gene non-coding region, and sum can reach 300 ten thousand (Brookes AJ.The essence of SNPs.Gene, 1999; 234:177-186.).SNPs is high with its density, and average every 1kb just has 1; Representative strong, be positioned at the inner SNPs of gene and possibly directly influence protein structure or expression level; Genetic stability is good, with microsatellite polymorphism comparatively speaking; Be easy to automated analysis; Because of SNPs is the allelotrope mark in the crowd; Can be simply with "+/-or 1/0 " direct somatotype; Become good genetic marker (Collins FS, Brooks LD, Chakravarti A.A DNA polymorphismdiscovery resource for research on human genetic variation.Genome Res.1998; 8:1229-1231).
The HRMA technology is a kind of novel method of gene type, and (polymerase chainreaction PCR) directly carries out HRMA after the amplification to sample, has realized the stopped pipe operation in the polymerase chain reaction.Because HRMA is based on the physical properties of nucleic acid fully and analyzes, and need not the sequence-specific probe, thereby HRMA detects the limitation that does not receive mutating alkali yl site and kind, needed just saturable dye of increase on conventional PCR basis.So, compare quantitative probe method, simplified running time and step, greatly reduce use cost.Thereby HRMA more and more receives publicity, and the use of saturated fluorescence dyestuff has simultaneously also significantly reduced the restraining effect of unsaturation optical dye to the PCR reaction.Sex change HPLC (denaturing highpressure liquidchromatography in HRMA and the classifying method in the past; DHPLC) and the thermograde capillary electrophoresis (temperature-gradient capillaryelectrophoresis, TGCE) identical.But both methods of back are for the very difficult somatotype of some wild-types and homozygous mutation.Although can mix with unknown sample through a known sample that isozygotys; Thereby form the allos chain and carry out somatotype, but can make experimentation repeat twice like this, distinguish assorted for the first time and with the variation of isozygotying; For the second time the known sample that isozygotys is mixed with unknown sample, thereby make work more loaded down with trivial details.Simultaneously, twice operation can not realize the stopped pipe operation, increases PCR product pollution problem.
Summary of the invention
The object of the present invention is to provide a kind of method of detection line plastochondria ND1 gene mononucleotide polymorphism.
It is the method and the specificity amplification primer of the SNP of mtDNA 3970 that a purpose more of the present invention is to provide detection line plastochondria ND1 site.
The 3rd purpose of the present invention is to provide the test kit of detection line plastochondria ND1 gene mononucleotide polymorphism and the application of this test kit.
For realizing above-mentioned purpose, the present invention adopts following concrete technical scheme:
The method of a kind of detection line plastochondria ND1 gene mononucleotide polymorphism of the present invention is characterized in that its concrete steps are following:
(1) extracts genomic dna;
(2) plastosome ND1 gene mononucleotide polymorphism identification:
Preparation mixed solution: genomic dna solution, PCR damping fluid, dNTP, Taq archaeal dna polymerase, primer, PLUS+ saturated fluorescence dyestuff, C1 and the C2 oligonucleotide confidential reference items and the pure water of step (1) preparation;
The PCR condition: 95 ℃ 5 minutes, 95 ℃ 1 minute, 56 ℃ 30 seconds, 72 ℃ 3 seconds, 72 ℃ 7 minutes, carry out 35 circulations; After reaction is accomplished, the PCR product is carried out the circulation of two sex change and renaturation again, 95 ℃ of sex change 30 seconds, 25 ℃ of renaturation 2 minutes, totally 2 circulations;
(3) sample with step (2) preparation carries out the analysis of high resolution melting curve.
The sample of step (2) preparation is checked order identification and checking high resolution melting curve somatotype result.
This order-checking is ordinary method, and giving birth to worker's biotechnology service company by Shanghai provides.This sequencing result has confirmed the accuracy of high resolution solubility curve analytical procedure of the present invention.
The source of extracting genomic dna in the aforesaid method can also can be histocyte for the body fluid that exsomatizes, and does not have specifically to limit.
The method of above-mentioned a kind of detection line plastochondria ND1 gene mononucleotide polymorphism, the method for wherein said extraction genomic dna is: will add 37 ℃ of incubated overnight of cell pyrolysis liquid except that the human peripheral of serum deprivation; Use the saturated phenol extraction DNA of Tris 2 times then; Collect the upper water item, add chloroform and the extracting of Virahol mixed solution 1 time; Collect water, add the ice absolute ethyl alcohol of 1/10 volume Glacial acetic acid min. 99.5 and 2.5 times of volumes ,-20 ℃ of sedimentation cell DNA that spend the night; Centrifugal then, add cold 70% ethanol centrifugal 1 time; The genomic dna of such acquisition is dissolved in the TE damping fluid, and the quantitatively determined mixture is in the specific absorption of 260nm then; DNA working fluid concentration is adjusted to 30ng/ul, puts-20 ℃ of refrigerators and preserves.
Above-mentioned PCR primer is:
The F1:5 '-CTAGTCTCAGGCTTCAACATC-3 ' of base sequence shown in SEQ ID No.1;
The R1:5 '-TGTTTGTGTATTCGGCTAT-3 ' of base sequence shown in SEQ ID No.2.
The above-mentioned detection method of the present invention can be used for detecting ND1 gene mtDNA 3970 site mutation situation.
The present invention also provides a kind of Auele Specific Primer that is used for detection line plastochondria ND1 gene mononucleotide polymorphism, and its length is 19～21bp, the F1:5 '-CTAGTCTCAGGCTTCAACATC-3 ' of base sequence shown in SEQ ID No.1; The R1:5 '-TGTTTGTGTATTCGGCTAT-3 ' of base sequence shown in SEQ ID No.2.Above-mentioned Auele Specific Primer can accurately amplify the ND1 gene order that contains mtDNA 3970 sites.
The test kit of a kind of detection line plastochondria ND1 gene mononucleotide polymorphism of the present invention, this test kit is formed, originated as follows by following reagent: test kit of the present invention supplies 10 person-portions to detect and uses-20 ℃ of preservations
(1) 108ul pure water (self-control);
(2) 20ul 10X PCR damping fluid (Takara);
(3) 4ul 10mM dNTP mixed solution (Pharmacia);
(4) 20ul 1unit/ul Taq archaeal dna polymerase (Takara);
(5) each 2ul 10pmol/ulF1 and R1 primer (self-control);
(6) 20ul 1XLCGreen PLUS+ saturated fluorescence dyestuff (Idaho);
(7) confidential reference items: each 1ul 10pmol/ul C1-1 (self-control) and C1-2 (self-control); Each 1ul 10pmol/ul C2-1 (self-control) and C2-2 (self-control);
Wherein primers F 1 base sequence is shown in SEQ ID No.1, and R1 is shown in SEQ ID No.2; Confidential reference items C1-1 is that the 3 ' end of base sequence shown in the SEQ ID No.3 adds 3 carbon alkyl modifications, and C1-2 is that the 3 ' end of base sequence shown in the SEQ ID No.4 adds 3 carbon alkyl modifications; C2-1 is that the 3 ' end of base sequence shown in the SEQ ID No.5 adds 3 carbon alkyl modifications, and C2-2 is that the 3 ' end of base sequence shown in the SEQ ID No.6 adds 3 carbon alkyl modifications.
3 ' end need be blocked in the confidential reference items (C1-1, C1-2, C2-1, C2-2), avoids its extension in pcr amplification.And 3 '-phosphorylation is used in 3 ' blocking-up usually, 2 ' 3 '-two deoxidation nuclear Nucleotide, 3 '-deoxynucleotide, 3 '-3 carbon alkyl.But 3 '-3 carbon alkyl, stability are better.
The method of use of test kit of the present invention:
1) through pcr amplification ND1 gene; Prepare mixed solution earlier; Add genomic dna solution 2ul, 2ul 10X PCR damping fluid, 0.4ul 10mM dNTP, 2ul Taq archaeal dna polymerase, F1 and the R1 of 0.2ul are sense primer and antisense primer respectively; 2ul LCGreen PLUS+ saturated fluorescence dyestuff, the C1 of 0.2ul and C2 are confidential reference items respectively.Then, add pure water, making TV is 20ul.Be reflected at 95 ℃ 5 minutes, 95 ℃ 1 minute, 56 ℃ 30 seconds, 72 ℃ 3 seconds, 72 ℃ 7 minutes, carry out 35 circulations.
2) after reaction is accomplished, the PCR product is carried out the circulation of two sex change and renaturation again, 95 ℃ of sex change 30 seconds, 25 ℃ of renaturation 2 minutes, totally 2 circulations.Then sample is carried out the melting curve analysis in Lightscanner TM HR-I 96 appearance.Be warmed up to 98 ℃ with the speed of 0.3C/ second from 50 ℃ through instrument, obtain the melting curve of sample.
3) polymorphum somatotype: melting curve per sample, if be T, fluorescent signal raises with temperature and at first begins to descend, if be C, fluorescent signal raises with temperature and then begins to descend.
The test kit of above-mentioned a kind of detection line plastochondria ND1 gene mononucleotide polymorphism can be used for predicting the application of old and feeble degenerative disease of being correlated with.When the detected result genotype was T, the experimenter was little with the degenerative disease relation relevant with aging; When if genotype is C, the experimenter has certain genetic affinity with old and feeble relevant degenerative disease, can make further detection or suitably note changing bad life style.
Above-mentioned and old and feeble relevant degenerative disease is: apoplexy, cardiovascular disorder, diabetes B, Parkinson's disease, senile dementia and/or cancer.
Design of primers of the present invention is human mitochondrial genom sequence (light chain) the Genbank:REFSEQ AC_000021.2 gi:115315570 that provides according to Cambridge reference sequences
(
Http:// www.ncbi.nlm.nih.gov/entrez/viewer.fcgi? Db=nucleotide& Val=11531 5570), be C at the 3970th, its antisense strand is the sudden change of G.
Measuring method of the present invention has been measured the genomic dna that derives from the people, sample not restriction as, body fluid (like blood, ascites and urine), histocyte (like hepatic tissue) etc. are through extracting and these samples of purifying can prepare genomic dna.
From genomic dna, can increase contains the dna fragmentation of ND1 transgenation point, with the great amount of samples that obtains to be used to measure.Thisly contain the sample that the dna fragmentation of ND1 transgenation point obtains, be particularly suitable for as measuring material through amplification.For example, can use primer to increase by PCR method, this primer contains the part of ND1 gene pleiomorphism through design rationally so that only increase.Auele Specific Primer of the present invention is one of inventive point of the present invention, and this primer those skilled in the art often regulation are equipped with the method preparation of primer.The base length in district to be amplified is unrestricted.When by preparation primer according to the invention, the working sample that can obtain to suit, it is as the dna fragmentation of amplification, and has specificity length.
The main principle of HRMA technology mainly is the formation according to the heterozygosis heteroduplex.Before the PCR reaction, add the saturated fluorescence dyestuff, in certain TR, pcr amplification product is carried out sex change, the dna double chain is unwind gradually, this moment, luminescent dye molecule came off from the dna double chain gradually, and fluorescent signal can descend.If certain individuality is a heterozygous mutant; Can form heterozygosis heteroduplex and unpaired base pair in this individual PCR product; This sample can at first unwind when temperature raises gradually so; Its fluorescent signal at first begins to descend, and the sample of the homozygous individual of this moment is higher owing to melting temperature(Tm), and it is slower that fluorescent signal does not descend or descends; The lightscanner instrument changes through the optical detection fluorescent signal and draws the temperature melting curve, accurately distinguishes wild-type, heterozygous mutant, pure and mild sudden change according to curve.
Carry out the high resolution melting curve when analyzing,, then need in the PCR reaction, add complementary double chain oligonucleotide above and below amplified fragments Tm value as confidential reference items for the error that temperature contrast in the process that reduces somatotype causes.3 ' end of this confidential reference items sequence need add 3 carbon alkyl to be modified, to avoid its extension in the PCR reaction.
Therefore, the present invention is according to the required dna fragmentation of PCR method amplification, and through above-mentioned reasonable primer, confidential reference items are able to design.The optical detection fluorescent signal changes generation temperature melting curve and confirms that they show different melting curves.According to the melting curve that in aforesaid method, obtains, can measure mtDNA C3970T SNP.
When carrying out gene test of the present invention; Preferably be used to measure reagent according to the mutation type existence of mtDNA C3970T SNP; Detection reagent comprises the particular agent as neccessary composition, and it is corresponding to the method that is used to measure mtDNA C3970T SNP mutation type.Specific reagent suitably selects by the measuring method that adopts.The characteristic of reagent is, composition measuring is necessary by the means of the mutation type of mtDNA C3970T SNP definition, as, dna fragmentation or high resolution melting curve are analyzed.Reagent; The primer that is used for the pcr amplification step of specific preparation for example; This step is used to comprise the specific amplified fragments of the catastrophe point of high resolution melting curve analysis list nucleotide polymorphisms, is not considered to the neccessary composition of diagnostic reagent of the present invention, and they also are contained among the diagnostic reagent of the present invention.
Advantage of the present invention and beneficial effect are:
In sum; The SNP method of detection ND1 provided by the present invention is a kind of biological detection method, compares with existing detection method; Has obvious improvement; No matter do not have special limitation like the inventive method for sample, be that body fluid and histocyte all can be used as the present invention and detect sample, makes easy to detect simple and direct; The Auele Specific Primer that the present invention designed is treated the base length of amplification region does not have strict demand, can obtain PCR product of the present invention and carry out the analysis of high resolution melting curve through Auele Specific Primer of the present invention; The present invention adds the saturated fluorescence dyestuff before the PCR reaction, the lightscanner instrument changes through the optical detection fluorescent signal and draws the temperature melting curve, can accurately distinguish wild-type, heterozygous mutant, pure and mild sudden change according to curve.The present invention is compared with prior art easy and simple to handle, detects with low costly, and detected result is accurate, has good using value and marketable value.
The present invention is a kind of biological gene locus detection method; With existing detection method significant effect is arranged relatively; Testing goal of the present invention is for learning mtDNA C3970T SNP; Its detected result can provide intermediate information for the degenerative disease prediction relevant with aging, but can not confirm or prediction and the old and feeble relevant ill foundation of degenerative disease as final, so the present invention does not belong to the diagnosis and the treat-ment of disease.
Foregoing has been explained technical scheme of the present invention and beneficial effect fully; Below in conjunction with accompanying drawing and embodiment the present invention is done further narration; So that the public has more deep understanding to summary of the invention; The embodiment of embodiment is best technical scheme, and is not limitation of the present invention.
Description of drawings
Fig. 1 is the gene type data plot of mtDNA C3970T; Left side figure is melting curve figure, and right figure is sample distribution figure.
Fig. 2 is polynucleotide sequence result identification plastosome ND1 gene mtDNA C3970T SNP mark after the information biology comparison of PCR-direct sequencing gained.
Fig. 3-1 and 3-2 are that the inventive method detected result is by PCR-direct sequencing proof diagram; Plastosome ND1 gene mtDNA 3970 gene locuss use this patent test kit genotyping result consistent with the dna sequencing result.
Embodiment
The english abbreviation that is used for the following example expression reagent is following.
When needing, sterilize with pressure kettle (120 ℃, 20 minutes)
EDTA: EDTA Disodium is commercially available
SDS: sodium lauryl sulphate is commercially available
TE damping fluid: 30ug/mlRNaseA, 10mmol/LTris-HCl pH8.0,1mmol/L EDTA 10PCR damping fluid: 100mM Tris-HCl (pH8.3), 500mM KCl, the self-control of 15mM magnesium chloride (MgCl2) 0.01% (W/V) gelatinum
DNTP: deoxynucleoside triphosphate is commercially available
Embodiment 1: the extraction of blood sample collection and genomic dna
One, sample is selected
The geographic long lived elder of door-to-door survey Guangxi crust horse, age >=90 year old.Realize clearly, life is supported oneself, and can cooperate inspection, no cardiovascular and cerebrovascular diseases, senile dementia, Parkinson's disease, the degeneration property disease that aging such as cancer is correlated with, totally 358 examples.Write down all experimenters' essential information and sign informed consent by me or relatives.This research has also obtained the approval of Ethics Committee of our unit.
Two, preparation genomic dna
In the presence of antithrombotics EDTA, at 2500rpm, spinning removed serum deprivation in 30 minutes with 5ml experimenter's peripheral blood of collecting.Then add 37 ℃ of incubated overnight of 5ml cell pyrolysis liquid (containing 10mmol/L Tris-HCl pH8.0,10mmol/L EDTApH8.0,15mmol/L Nacl, 0.4%SDS, 0.1mg/ml Proteinase K).Use then the saturated phenol of Tris (pH8.0) 5ml extract DNA 2 times (12000r/min, 10min).Collect the upper water item, add 5ml chloroform and Virahol (24: 1) mixed solution extracting 1 time (centrifugal 12000r/min, 10min).Collect the water item, add the ice absolute ethyl alcohol of 1/10 volume 3M Glacial acetic acid min. 99.5 and 2.5 times of volumes ,-20 ℃ of sedimentation cell DNA that spend the night.Centrifugal then 12000r/min, 30min, add cold 70% ethanol 1ml wash 1 time (centrifugal 12000r/min, 10min).The genomic dna of such acquisition is dissolved in the TE damping fluid, and the quantitatively determined mixture is in the specific absorption of 260nm then.DNA working fluid concentration is adjusted to 30ng/ul, puts-20 ℃ of refrigerators and preserves.
The identification of embodiment 2:SNP is confirmed
The present invention adopts HRMA and PCR sequencing technologies simultaneously C3970T site (its loci is to being A/G) to be detected.
One. Auele Specific Primer is following:
F1：5’-CTAGTCTCAGGCTTCAACATC-3’(SEQ?ID?NO.1)
R1：5’-TGTTTGTGTATTCGGCTAT-3’(SEQ?ID?NO.2)
Two. through near the part fragment pcr amplification C3970T; Preparation mixed solution: add the foregoing description 1 preparation genomic dna solution 2ul, 2ul 10X PCR damping fluid, 0.4ul 10mM dNTP, 2ul Taq archaeal dna polymerase, difference 0.2ul above-mentioned steps 1) every kind of primer of narration; 2ul LCGreen PLUS+ saturated fluorescence dyestuff, C1 and the C2 oligonucleotide confidential reference items of 0.1ul respectively.Then, add pure water, making TV is 20ul.Be reflected at 95 ℃ 5 minutes, 95 ℃ 1 minute, 56 ℃ 30 seconds, 72 ℃ of 3s, 2 ℃ 7 minutes, carry out 35 circulations.After reaction is accomplished, the PCR product is carried out the circulation of two sex change and renaturation again, 95 ℃ of sex change 30 seconds, 25 ℃ of renaturation 2 minutes, totally 2 circulations.
Three. through sample is carried out the melting curve analysis in Lightscanner TM HR-I 96 appearance.Instrument is warmed up to 98 ℃, the melting curve of acquisition sample with the speed of 0.3C/ second from 50 ℃.As shown in Figure 1, Fig. 1 is the gene type data plot of mtDNA C3970T; Left side figure is melting curve figure, and right figure is sample distribution figure, melting curve per sample, if be T, fluorescent signal raises with temperature and at first begins to descend, and if be C, fluorescent signal raises with temperature and then begins to descend.Curve is the different representations of same sample with grid in the collection of illustrative plates; Black curve and grid are represented mtDNA 3970T; Grey curve and grid are represented as mtDNA 3970C; S in the grid represents the positive contrast of this sample.
The foregoing description result verification:
Utilize the PCR-direct sequencing that the sample of embodiment 1 preparation is carried out dna sequencing, identification and checking embodiment 2 high resolution melting curve somatotype results.This order-checking is not given unnecessary details at this for ordinary method, and this order-checking is given birth to worker's biotechnology service company by Shanghai and provided.This sequencing result has confirmed the accuracy of high resolution solubility curve analytical procedure of the present invention.As shown in Figure 2, be polynucleotide sequence result identification plastosome ND1 gene mtDNA C3970T SNP mark after the information biology comparison of PCR-direct sequencing gained; With shown in the 3-2, Fig. 3-1 and 3-2 are that the inventive method detected result is by PCR-direct sequencing proof diagram like Fig. 3-1; Plastosome ND1 gene mtDNA 3970 gene locuss use this patent test kit genotyping result consistent with the dna sequencing result.
Embodiment 3:mtDNA 3970 SNPs are relevant with the degenerative disease relevant with aging
One. statistical method: utilize carrier's frequency of Yates chi square test calculating mtDNA 3970 SNPs in STATA8.0 and the SPSS11.0 software, carry out continuous correction and one-sided asymptotic probability analysis, statistical significance level is set at P＜0.05.Adopt single factor Logistic regression analysis to calculate long-lived risk OR value and 95% credibility interval (CI) thereof.
Two. the result
1. healthy subjects mtDNA 3970 SNPs distribute
Measured the gene pleiomorphism of 362 healthy subjects (no cardiovascular and cerebrovascular diseases, cancer etc., age≤60 year old) by the method for embodiment 1 and 2.285 people have C polymorphum (78.1%) at the 3970th bit base, and 77 people have the polymorphum (21.3%) of T.
2. good health and a long life old man (do not have and old and feeble relevant degenerative disease person, age >=90 year old) mtDNA 3970 SNPs distribution.
The method of pressing embodiment 1 and 2 is measured an above-mentioned good health and a long life old man gene pleiomorphism.249 people have C nucleotide polymorphisms (69.5%) at the 3970th, find that 109 people have T nucleotide polymorphisms (30.5%).
3. good health and a long life old man group and healthy subjects control group are relatively
The frequency distribution of healthier long lived elder group and normal healthy controls group mtDNA 3970 SNPs sees table 1 for details.
Table 1mtDNA 3970 SNPs (SNP) frequency risk exists
The comparison of good health and a long life old man group and healthy subjects
Visible by table 1, the C loci in the common SNP site that mtDNA is the 3970th is the G loci on its DNA complementary strand promptly; When sporting T; Distribution frequency in good health and a long life old man colony is much higher than the frequency in the control population, differs about 9%, and significance difference (P=0.005) is arranged.And the frequency of OR value reflection site T exceeds 1.6 times of normal peoples in the healthy old men crowd, and 95%CI lower limit＞1 shows that all this is a relevant allelotrope of the degenerative disease relevant with aging.C3970T is positioned at the ND1 gene; Its expression product is nadh dehydrogenase (complex body I) subunit 1; This sports same sense mutation; This sudden change possibly be the degenerative disease correlated inheritance mark relevant with aging, also possibly influence transcribing of DNA etc. through influencing sequence and the space structure of DNA on function.
Embodiment 4: detection kit
Test kit of the present invention supplies 10 person-portions to detect application, and storage temperature is-20C, comprises:
The 108ul pure water,
20ul 10X PCR damping fluid,
4ul 10mM dNTP mixed solution,
20ul (1unit/ul) Taq archaeal dna polymerase,
Each 2ul (10pmol/ul) F1 (SEQ ID No.1) and R1 (SEQ ID No.2) primer,
20ul 1XLCGreen PLUS+ saturated fluorescence dyestuff,
Each 1ul (10pmol/ul) C1-1 (SEQ ID No.4) and C1-2 (SEQ ID No.5),
Each 1ul (10pmol/ul) C2-1 (SEQ ID No.6) and C2-2 (SEQ ID No.7).
Primer sequence is:
F1：5’-CTAGTCTCAGGCTTCAACATC-3’(SEQ?ID?NO：1)
R1：5’-TGTTTGTGTATTCGGCTAT-3’(SEQ?ID?NO：2)
The confidential reference items sequence is:
C1-1：(SEQ?ID?NO：3)
5’-TTAAATTATAAAATATTTATAATATTAATTATATATATATAAATATAATA-C3-3’
C1-2：(SEQ?ID?NO：4)
5’-TATTATATTTATATATATATAATTAATATTATAAATATTTTATAATTTAA-C3-3’
C2-1：(SEQ?ID?NO：5)
5’-GCGCGGCCGGCACTGACCCGAGACTCTGAGCGGCTGCTGGAGGTGCGGAAGCGGAGGGGCGGG-C3-3’
C2-2：(SEQ?ID?NO：6)
5’-CCCGCCCCTCCGCTTCCGCACCTCCAGCAGCCGCTCAGAGTCTCGGGTCAGTGCCGGCCGCGC-C3-3’
This test kit can detect the SNP of C → T of the 3970th easily after PCR-HRMA detects.
The present invention has the illustration of practicality:
1. the detection method of mtDNA 3970 SNPs of the present invention; The T loci that can be used for the common SNP on the mitochondrial ND1 gene of analyst; Whether the degenerative disease relevant with aging has relation to investigate to individuality in application, is beneficial to carry out the improvement of early intervention and life style.
2. utilize the present invention to set forth mtDNA 3970 nucleotide variations, as one of biomarker, the screening of useful as drug designed molecules target promotes old relative disease new drug development.
3. the nucleotide sequence and the related locus of detection mtDNA 3970 SNPs set up of the present invention, but highly sensitive is applied to the test kit that longevity gene detects usefulness specifically.
As stated, reach a conclusion, the mtDNA SNP is in the polymorphum and old and feeble relevant degenerative disease tool significant correlation property of the 3970th bit base.
The present invention has narrated the relevant new mutant point of the degenerative disease relevant with aging, and a kind of method of the mtDNA of mensuration 3970 SNPs is provided.And, according to the present invention, only need a small amount of DNA sample just to be enough to measure the polymorphum of mtDNA 3970 SNPs.
Estimation according to dead level before and after nineteen ninety shows, the live to be hundred average probability of above longevity of 80 years old old man is respectively 0.65%.The so rare people longevity that might live to be hundred; Prove absolutely that with common people's faciation ratio, long lived elder is beneficial to aged healthy gene than carrying; But represent the sampling results of 60,000 colonies approximately according to probability calculation knowledge capital experiment institute sampling results; Have feasibility and accuracy, the coincidence statistics standard can be used for other crowds' of other areas detection.
Sequence table
< 110>Beijing Hospital
< 120>method, test kit and the application thereof of detection line plastochondria ND1 gene mononucleotide polymorphism
<130>
<160>6
<170>PatentIn?version?3.5
<210>1
<211>21
<212>DNA
< 213>forward primer artificial sequence
<400>1
ctagtctcag?gcttcaacat?c 21
<210>2
<211>19
<212>DNA
< 213>reverse primer artificial sequence
<400>2
tgtttgtgta?ttcggctat 19
<210>3
<211>50
<212>DNA
< 213>confidential reference items C1-1
<400>3
ttaaattata?aaatatttat?aatattaatt?atatatatat?aaatataata 50
<210>4
<211>50
<212>DNA
< 213>confidential reference items C1-2
<400>4
tattatattt?atatatatat?aattaatatt?ataaatattt?tataatttaa 50
<210>5
<211>63
<212>DNA
< 213>confidential reference items 2-1
<400>5
gcgcggccgg?cactgacccg?agactctgag?cggctgctgg?aggtgcggaa?gcggaggggc 60
ggg 63
<210>6
<211>63
<212>DNA
< 213>confidential reference items 2-2
<400>6
cccgcccctc?cgcttccgca?cctccagcag?ccgctcagag?tctcgggtca?gtgccggccg 60
cgc 63
Claims (3)
1. the test kit of a detection line plastochondria ND1 gene mononucleotide polymorphism is characterized in that this test kit is made up of following reagent:
(1) 108 μ l pure water;
(2) 20 μ l 10X PCR damping fluids;
(3) 4 μ l 10mM dNTP mixed solutions;
(4) 20 μ l lunit/ μ l Taq archaeal dna polymerases;
(5) each 2 μ l 10pmol/ μ l F1 and R1 primer;
(6) 20 μ l 1XLCGreen PLUS+ saturated fluorescence dyestuffs,
(7) confidential reference items: each 1 μ l 10pmol/ μ l C1-1 and C1-2; Each 1 μ l 10pmol/ μ l C2-1 and C2-2;
Wherein primers F 1 base sequence is shown in SEQ ID No.1, and R1 is shown in SEQ ID No.2; Confidential reference items C1-1 is that the 3 ' end of base sequence shown in the SEQ ID No.3 adds 3 carbon alkyl modifications, and C1-2 is a base sequence shown in the SEQ ID No.4
Show that desktop (2) .1nk3 ' end adds 3 carbon alkyl and modifies; C2-1 is that the 3 ' end of base sequence shown in the SEQ ID No.5 adds 3 carbon alkyl modifications, and C2-2 is that the 3 ' end of base sequence shown in the SEQ ID No.6 adds 3 carbon alkyl modifications.
2. an Auele Specific Primer that is used for detection line plastochondria ND1 gene mononucleotide polymorphism is characterized in that, its length is 19～21bp,
The F1:5 '-CTAGTCTCAGGCTTCAACATC-3 ' of base sequence shown in SEQ ID No.1;
The R1:5 '-TGTTTGTGTATTCGGCTAT-3 ' of base sequence shown in SEQ ID No.2.
3. a kind of Auele Specific Primer that is used for detection line plastochondria ND1 gene mononucleotide polymorphism according to claim 2 is characterized in that described Auele Specific Primer can amplify the ND1 gene order that contains mtDNA 3970 sites.
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| CN2009100763670A CN101775435B (en) | 2009-01-14 | 2009-01-14 | Method and kit for detecting polymorphism of mitochondrial ND1 gene mononucleotide, and application of kit |
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| CN104928373B (en) * | 2015-06-02 | 2017-10-03 | 江苏雄鸣医药科技有限公司 | A kind of gene diagnosis kit of Parkinson's |
| CN110016496A (en) * | 2019-03-22 | 2019-07-16 | 大连医科大学 | Utilize the method for fluorescence quantitative PCR method detection FUT2 gene type |
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Non-Patent Citations (3)
| Title |
|---|
| 于君,等.线粒体DNA突变和疾病的关系研究进展.《中国全科医学》.2008,第11卷(第12B期),2273－2276. * |
| 孔 放,等.人类长寿相关基因研究进展.《遗 传》.2006,第28卷(第7期),874-879. * |
| 杨 泽，等.衰老与长寿的遗传机制探讨.《国外医学遗传学分册》.2000,第23卷(第4期),211-214. * |
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