CN101525665A - SNP G10320A molecular marker of mitochondrion ND3 gene of senescence-associated degenerative disease, detecting method and kit thereof - Google Patents
SNP G10320A molecular marker of mitochondrion ND3 gene of senescence-associated degenerative disease, detecting method and kit thereof Download PDFInfo
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Abstract
The present invention discloses a molecular marker of senescence-associated degenerative disease, a method for detecting the molecular marker SNP and a detecting kit thereof. The method of the invention has no special restriction to the sample. The body fluid and histiocyte can be equally taken as the detecting sample of the invention to cause that the detection is convenient and concise. The specific primer designed by the invention has no strict requirement for the length of basic group in the amplification area. The PCR product of the invention can be obtained through the specific primer for analyzing the high resolution fluxing curve. The saturated fluorescent dye is added before PCR reaction. A light-scanner detects the variation of fluorescent signal through optical detection and draws a temperature fluxing curve. The wild type, heterozygous mutation and homozygous mutation can be accurately differentiated according to the curve. The invention has the advantages of easy operation, low detection cost, accurate detection result, excellent application value and market value.
Description
Technical field
The present invention relates to the method for a kind of detection chondriosome ND 3 gene mononucleotide polymorphism (SNP), the method for specifically a kind of SNP that detects chondriosome ND 3 gene mtDNA 10320 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 enter aging society, the aged accounts for total man's mouth constituent ratio (10%) and is increasing year by year, expect the year two thousand twenty, 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.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 is subjected to 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 by reducing old and feeble relevant degenerative disease or delaying senility.But the long-lived definite factor of influence is not illustrated 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 studies have shown that inherited genetic factors plays an important role in the mechanism of longevity.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 may play important effect in the mechanism of longevity.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 concerns the concern that more and more is being subjected to the investigator with old and feeble relevant degenerative disease.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.As 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: as 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 be converted to T (thymus pyrimidine) by spontaneous deaminizating, and SNPs has comprised the 80-90% of known polymorphism, 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 with its density height, and average every 1kb just has 1; Representative strong, the SNPs that is positioned at gene inside may 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 is not subjected to mutating alkali yl site and kind, needed just saturable dye of increase on conventional PCR basis.So, compare quantitative probe method, simplified operating 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 fluorescence dye to the PCR reaction.Sex change high pressure liquid chromatography (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 by 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 the chondriosome ND 3 gene molecule marker of a kind of and old and feeble relevant degenerative disease and detect this molecular marker method.
Another object of the present invention is to provide detection chondriosome ND 3 site is the method and the specificity amplification primer of the single nucleotide polymorphism of mtDNA 10320.
A further object of the present invention is to provide the test kit of detection chondriosome ND 3 gene SNP G10320A molecule marker and the application of this test kit.
For achieving the above object, the present invention adopts following concrete technical scheme:
A kind of chondriosome ND 3 gene SNP G10320A molecule marker that detects the degenerative disease relevant with aging, its dna sequence dna is shown in SEQ ID No.1.
Above-mentioned detection and old and feeble relevant degenerative disease chondriosome ND 3 gene SNP G10320A molecule marker, wherein, there is a molecular marker SNP G10320A in described SEQ ID No.10320 site.
The above-mentioned degenerative disease chondriosome ND 3 gene SNP G10320A molecule marker relevant with aging, wherein, the 10320th site molecule marker of described SEQ ID No.1 is G or A.
A kind of detection chondriosome ND 3 gene SNP G10320A molecular marker method of the present invention is characterized in that its concrete steps are as follows:
(1) extracts genomic dna;
(2) chondriosome ND 3 gene single nucleotide polymorphism identification:
Preparation mixed solution: genomic dna solution, PCR damping fluid, dNTP, Taq archaeal dna polymerase, primer, PLUS+ saturated fluorescence dyestuff, C1 oligonucleotide probe and the pure water of step (1) preparation;
The PCR condition: 95 ℃ 5 minutes, 95 ℃ 1 minute, 63.5 ℃ 30 seconds, 72 ℃ 10 seconds, 72 ℃ 7 minutes, carry out 35 circulations; After reaction is finished, 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.
Above-mentioned a kind of detection chondriosome ND 3 gene SNP G10320A molecular marker method, the method for wherein said extraction genomic dna is: will add 37 ℃ of overnight incubation 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 the water item, add the ice dehydrated alcohol of 1/10 volume Glacial acetic acid 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 quantitative assay 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 '-ATTTGATCTAGAAATTGCCCTCC-3 ' of base sequence shown in SEQ ID No.2;
The R1:5 '-TTGTAGTCACTCATAGGCCAG-3 ' of base sequence shown in SEQ ID No.3.
The above-mentioned detection method of the present invention can be used for detecting ND3 gene mtDNA 10320 site mutation situations.
The present invention also provides a kind of Auele Specific Primer that is used to detect chondriosome ND 3 gene SNP G10320A molecule marker, its length is 21～23bp, and base sequence is shown in SEQ ID No.2: F1:5 '-ATTTGATCTAGAAATTGCCCTCC-3 '; The R1:5 '-TTGTAGTCACTCATAGGCCAG-3 ' of base sequence shown in SEQ ID No.3.Above-mentioned Auele Specific Primer can accurately amplify the ND3 gene order that contains mtDNA 10320 sites.
A kind of test kit that detects chondriosome ND 3 gene SNP G10320A molecule marker of the present invention, this test kit is formed, originates as follows by following reagent: test kit of the present invention detects for 10 person-portions and uses-20 ℃ of preservations
(1) 130ul 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) 2ul 1pmol/ulF1 primer (self-control);
(6) 2ul 10pmol/ulR1 primer (self-control)
(6) 20ul 1XLCGreen PLUS+ saturated fluorescence dyestuff (Idaho);
(7) probe: 2ul 10pmol/ul C1 (self-control);
Wherein primers F 1 base sequence is shown in SEQ ID No.2, and R1 is shown in SEQ ID No.3; Probe C1 base sequence is shown in SEQ ID No.4.
5’-ACAACTAACCTACCACTAATAGTTATGTCATCC-C3-3’
3 ' end need be blocked in this probe, 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 using method of test kit of the present invention:
1) by pcr amplification ND3 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 probe of 0.2ul.Then, add pure water, making cumulative volume is 20ul.Be reflected at 95 ℃ 5 minutes, 95 ℃ 1 minute, 63.5 ℃ 30 seconds, 72 ℃ 10 seconds, 72 ℃ 7 minutes, carry out 35 circulations.
2) after reaction is finished, 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 instrument.Be warmed up to 98 ℃ with the speed of 0.3C/ second from 50 ℃ by instrument, obtain the melting curve of sample.
3) polymorphism somatotype: melting curve per sample, if be A, fluorescent signal raises with temperature and at first begins to descend, if be G, fluorescent signal raises with temperature and then begins to descend.
Above-mentioned a kind of test kit that detects the chondriosome ND 3 gene mononucleotide polymorphism can be used for predicting the application of old and feeble degenerative disease of being correlated with.When detected result SNP G10320A molecular marker gene type was A, the experimenter was little with old and feeble relevant degenerative disease relation; When if genotype is G, the experimenter has certain genetic affinity with old and feeble relevant degenerative disease, can make further detection or suitably note changing bad living habit.
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 be 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﹠amp; Val=11531 5570), be G at the 10320th, its antisense strand is the sudden change of A.
Measuring method of the present invention has been measured the genomic dna that derives from the people, sample without limits as, body fluid (as blood, ascites and urine), histocyte (as hepatic tissue) etc. are by extracting and these samples of purifying can prepare genomic dna.
From genomic dna, can increase contains the dna fragmentation of ND3 transgenation point, with the great amount of samples that obtains to be used to measure.Thisly contain the sample that the dna fragmentation of ND3 transgenation point obtains, be particularly suitable for as measuring material by amplification.For example, can use primer to increase by PCR method, this primer contains the part of ND3 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 of the present 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.Add the saturated fluorescence dyestuff before the PCR reaction, in certain temperature range 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 because melting temperature(Tm) is higher, it is slower that fluorescent signal does not descend or descends, the lightscanner instrument changes by 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.
When carrying out the analysis of high resolution melting curve, adopt unmarked probe method promptly to utilize the primer of inequality to carry out the asymmetric pcr reaction, in the PCR product except double-stranded DNA, also there is an excessive single stranded DNA with probe hybridization, by in certain temperature range, carrying out sex change, make the probe with single stranded DNA incomplete pairing (formation heteroduplex) unwind earlier, the principle of unwinding behind the paired probe is fully carried out the SNPs somatotype of probe area.This method can overcome the limitation of in the past little amplicon method, with two kinds of accurate somatotypes of homozygote.3 ' end of this probe 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 by above-mentioned reasonable primer, probe is designed.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 G10320A single nucleotide polymorphism.
When carrying out gene test of the present invention, the preferred reagent that is used to measure according to the mutation type existence of mtDNA G10320A single nucleotide polymorphism, detection reagent comprises the particular agent as neccessary composition, and it is corresponding to the method that is used to measure mtDNA G10320A single nucleotide polymorphism mutation type.Specific reagent is suitably selected by the measuring method that adopts.The feature of reagent is, composition measuring is necessary by the means of the mutation type of mtDNA G10320A single nucleotide polymorphism 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:
The present invention proposes a kind of new chondriosome ND 3 gene SNP G10320A molecule marker and can be used as detection and old and feeble relevant degenerative disease application.The SNP method of detection ND3 provided by the present invention, be a kind of biological detection method, compare with existing detection method, has obvious improvement, there is not special restriction as the inventive method for sample, no matter be that body fluid and histocyte all can be used as the present invention and detect sample, make easy to detect simple and direct; The designed Auele Specific Primer of the present invention 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 by Auele Specific Primer of the present invention; The present invention adds the saturated fluorescence dyestuff before the PCR reaction, the lightscanner instrument changes by 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 G10320A single nucleotide polymorphism, its detected result can provide intermediate information for the degenerative disease prediction relevant with aging, this detection information can assist the person under inspection to note diet and the living habit of setting up normal health, but can not determine or prediction and the old and feeble relevant ill foundation of degenerative disease as final, so the present invention is not the diagnosis and the methods of treatment of disease.
Foregoing has illustrated technical scheme of the present invention and beneficial effect fully, below in conjunction with the drawings and specific embodiments 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 G10320A; Show among the figure that different genotype forms different melting curves with temperature change, black curve is genotype A, and gray curve is genotype G.
Fig. 2 is polynucleotide sequence result identification plastosome ND1 gene mtDNA G10320A 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; Chondriosome ND 3 gene mtDNA G10320A gene locus uses 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 as follows.
When needing, with pressure kettle (120 ℃, 20 minutes) sterilization
EDTA: disodium ethylene diamine tetraacetate 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 disease that aging such as cancer is correlated with, totally 372 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, centrifugation removed serum deprivation in 30 minutes with 5ml experimenter's peripheral blood of collecting.Then add 37 ℃ of overnight incubation 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 dehydrated alcohol of 1/10 volume 3M Glacial acetic acid 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 quantitative assay 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 determined
The present invention adopts HRMA and PCR sequencing technologies simultaneously G10320A site (its loci is to being C/T) to be detected.
One. Auele Specific Primer is as follows:
F1：5’-ATTTGATCTAGAAATTGCCCTCC-3’；(SEQ?ID?NO.2)
R1：5’-TTGTAGTCACTCATAGGCCAG-3’(SEQ?ID?NO.3)
Two. by near the part fragment pcr amplification G10320A; The preparation mixed solution: adding the foregoing description 1 prepares 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 probe of 0.2ul.Then, add pure water, making cumulative volume is 20ul.Be reflected at 95 ℃ 5 minutes, 95 ℃ 1 minute, 63.5 ℃ 30 seconds, 72 ℃ 10 seconds, 72 ℃ 7 minutes, carry out 35 circulations.After reaction is finished, 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. by sample is carried out the melting curve analysis in Lightscanner TM HR-I 96 instrument.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 G10320A; Melting curve per sample, if be A, fluorescent signal raises with temperature and at first begins to descend, if be G, fluorescent signal raises with temperature and then begins to descend.Black curve is represented mtDNA10320A in the collection of illustrative plates; The grey curve is represented as mtDNA 10320G.
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, the polynucleotide sequence result for PCR-direct sequencing gained discerns chondriosome ND 3 gene mtDNA G10320A SNP mark after the information biology comparison; Shown in Fig. 3-1 and 3-2, Fig. 3-1 and 3-2 are that the inventive method detected result is by PCR-direct sequencing proof diagram; Chondriosome ND 3 gene mtDNA 10320 gene locuss use this patent test kit genotyping result consistent with the dna sequencing result.
Embodiment 3:mtDNA 10320 single nucleotide polymorphism are relevant with the degenerative disease relevant with aging
One. statistical method: carrier's frequency of utilizing Pearson chi square test calculating mtDNA 10320 single nucleotide polymorphism 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 people mtDNA 10320 single nucleotide polymorphism distribute
Measured 384 healthy people's (age≤60 year old) gene pleiomorphism by the method for embodiment 1 and 2.379 people have G polymorphism (98.7%) at the 10320th bit base, and 5 people have the polymorphism (1.3%) of A.
2. healthy old men (do not have and old and feeble relevant degenerative disease person, age 〉=90 year old) mtDNA 10320 single nucleotide polymorphism distribution
The method of pressing embodiment 1 and 2 is measured an above-mentioned old man gene pleiomorphism.359 people have G nucleotide polymorphisms (96.5%) at the 10320th, find that 13 people have A nucleotide polymorphisms (3.5%).
3. healthy old men group and normal healthy controls group are relatively
The frequency distribution of healthier old man's group and normal healthy controls group mtDNA 10320 single nucleotide polymorphism sees table 1 for details.
The comparison of table 1 mtDNA 10320 single nucleotide polymorphism (SNP) frequency risk in healthy old men group and healthy normal population
By table 1 as seen, the G loci in the common SNP site that mtDNA is the 10320th is the C loci on its DNA complementary strand promptly, when sporting A, distribution frequency in healthy old men colony is much higher than the frequency in the control population, differs about 2.2%, and significance difference (P=0.048) is arranged.And the frequency of OR value reflection site A exceeds 2.745 times of normal peoples in the healthy old men crowd, shows that all this is and a healthy old and feeble relevant allelotrope.G10320A is positioned at the ND3 gene, its expression product is nadh dehydrogenase (complex body I) subunit 3, and this sports missense mutation, makes the Xie Ansuan of coding become Isoleucine, may on function, pass through to influence the function of proteins encoded, thereby influence mitochondrial function.
Embodiment 4: detection kit
Test kit of the present invention detects for 10 person-portions and uses, and storage temperature is-20 ℃, comprising:
The 130ul pure water;
20ul 10X PCR damping fluid;
4ul 10mM dNTP mixed solution;
20ul 1unit/ul Taq archaeal dna polymerase;
2ul 1pmol/ulF1 primer (SEQ ID No.1);
2ul 10pmol/ulR1 primer (SEQ ID No.2);
20ul 1XLCGreen PLUS+ saturated fluorescence dyestuff;
Probe: 2ul 10pmol/ul C1 (SEQ ID No.3).
Primer sequence is:
F1：5’-ATTTGATCTAGAAATTGCCCTCC-3’；(SEQ?ID?No.2)
R1：5’-TTGTAGTCACTCATAGGCCAG-3’(SEQ?ID?No.3)
Probe sequence is:
C1：(SEQ?ID?No.4)
5’-ACAACTAACCTACCACTAATAGTTATGTCATCC-C3-3’
This test kit can detect the SNP of G → A of the 10320th easily after PCR-HRMA detects.
The present invention has the illustration of practicality:
1. the detection method of mtDNA 10320 single nucleotide polymorphism of the present invention, the A loci that can be used for the common SNP on the mitochondrial ND3 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 living habit.
2. utilize the present invention to set forth mtDNA 10320 nucleotide variations, as one of biomarker, the screening of the molecular target of useful as drug design promotes old relative disease new drug development.
3. the nucleotide sequence and the related locus of detection mtDNA 10320 single nucleotide polymorphism set up of the present invention, but highly sensitive is applied to the test kit that longevity gene detects usefulness specifically.
As mentioned above, reach a conclusion, the mtDNA single nucleotide polymorphism is in the polymorphism and old and feeble relevant degenerative disease tool significant correlation of the 10320th 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 10320 single nucleotide polymorphism is provided.And, according to the present invention, only need a small amount of DNA sample just to be enough to measure the polymorphism of mtDNA 10320 single nucleotide polymorphism.
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, with common people's faciation ratio, long lived elder is beneficial to aged healthy gene than carrying, represent the sampling results of about 60,000 colonies according to probability calculation this experiment as can be known 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〉chondriosome ND 3 gene SNP G10320A molecule marker, the detection method with senescence-associated degenerative disease reaches
Test kit
<130>
<160>4
<170>PatentIn?version?3.5
<210>1
<211>133
<212>DNA
＜213〉human mitochondrial gene
<400>1
atttgatcta?gaaattgccc?tccttttacc?cctaccatga?gccctacaaa?caactaacct 60
gccactaata?rttatgtcat?ccctcttatt?aatcatcatc?ctagccctaa?gtctggccta 120
tgagtgacta?caa 133
<210>2
<211>23
<212>DNA
＜213〉artificial sequence
<400>2
atttgatcta?gaaattgccc?tcc 23
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<400>3
ttgtagtcac?tcataggcca?g 21
<210>4
<211>33
<212>DNA
＜213〉artificial sequence
<400>4
acaactaacc?taccactaat?agttatgtca?tcc 33
Claims (10)
1, a kind of and old and feeble relevant degenerative disease chondriosome ND 3 gene SNP G10320A molecule marker is characterized in that dna sequence dna is shown in SEQ ID No.1.
2, according to claim 1 and old and feeble relevant degenerative disease chondriosome ND 3 gene SNP G10320A molecule marker is characterized in that there is a molecular marker SNP G10320A in the 10320th site among the described SEQ ID No.1.
3, according to claim 2 and old and feeble relevant degenerative disease chondriosome ND 3 gene SNP G10320A molecule marker is characterized in that the 10320th site molecule marker of described SEQ ID No.1 is G or A.
4, a kind of test right requires 2 described chondriosome ND 3 gene SNP G10320A molecular marker method, it is characterized in that its concrete steps are as follows:
(1) extracts genomic dna;
(2) chondriosome ND 3 gene single nucleotide polymorphism identification:
Preparation mixed solution: genomic dna solution, PCR damping fluid, dNTP, Taq archaeal dna polymerase, PCR primer, PLUS+ saturated fluorescence dyestuff, oligonucleotide probe and the pure water of step (1) preparation;
The PCR condition: 95 ℃ 5 minutes, 95 ℃ 1 minute, 63.5 ℃ 30 seconds, 72 ℃ 10 seconds, 72 ℃ 7 minutes, carry out 35 circulations; After reaction is finished, 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.
5, detection chondriosome ND 3 gene SNP G10320A molecular marker method according to claim 4 is characterized in that described PCR primer is:
Base sequence is shown in the SEQ ID No.2:
F1：5’-ATTTGATCTAGAAATTGCCCTCC-3’；
Base sequence is shown in the SEQ ID No.3:
R1：5’-TrGTAGTCACTCATAGGCCAG-3’。
6, a kind ofly be used for the Auele Specific Primer that test right requires 2 described chondriosome ND 3 gene SNP G10320A molecule markers, it is characterized in that its length is 21～23bp, base sequence is shown in SEQ ID No.2
F1:5 '-ATTTGATCTAGAAATTGCCCTCC-3 '; The R1:5 '-TTGTAGTCACTCATAGGCCAG-3 ' of base sequence shown in SEQ ID No.3.
7, Auele Specific Primer according to claim 6 is characterized in that, described Auele Specific Primer can amplify the ND3 gene order that contains the mtDNA10320 site.
8, a kind of test right requires the test kit of 2 described chondriosome ND 3 gene SNP G10320A molecule markers, it is characterized in that, this test kit is made up of following reagent:
(1) 130ul pure water;
(2) 20ul 10X PCR damping fluid;
(3) 4ul 10mM dNTP mixed solution;
(4) 20ul 1unit/ul Taq archaeal dna polymerase;
(5) 2ul 1pmol/ul F1 primer;
(6) 2ul 10pmol/ul R1 primer;
(7) 20ul 1XLCGreen PLUS+ saturated fluorescence dyestuff,
(8) probe: 2ul 10pmol/ul C1;
Wherein primers F 1 base sequence is shown in SEQ ID No.2, and R1 is shown in SEQ ID No.3; Probe C1 base sequence is shown in SEQ ID No.4.
9, the test kit of the described detection chondriosome ND 3 gene SNP of claim 8 G10320A molecule marker is used to predict the application of old and feeble degenerative disease of being correlated with.
10, the application of the test kit of detection chondriosome ND 3 gene SNP G10320A molecule marker according to claim 9, it is characterized in that the degenerative disease that described aging is relevant is: apoplexy, cardiovascular disorder, diabetes B, Parkinson's disease and/or senile dementia.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409088A (en) * | 2011-09-22 | 2012-04-11 | 郭奇伟 | Method for detecting gene copy number variation |
| CN102766683A (en) * | 2011-05-02 | 2012-11-07 | 爱科来株式会社 | Gene mutation detection probe |
| CN103013989A (en) * | 2011-09-23 | 2013-04-03 | 深圳北京大学香港科技大学医学中心 | Single-nucleotide polymorphisms of cyclin dependent kinase 5 related to senile dementia incidence |
| CN111676281A (en) * | 2019-12-31 | 2020-09-18 | 安徽医科大学第一附属医院 | Kit and method for detecting human mitochondrial ND3 gene 10191 locus genotype based on HRM method |
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2009
- 2009-04-13 CN CN200910081562A patent/CN101525665A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102766683A (en) * | 2011-05-02 | 2012-11-07 | 爱科来株式会社 | Gene mutation detection probe |
| CN102409088A (en) * | 2011-09-22 | 2012-04-11 | 郭奇伟 | Method for detecting gene copy number variation |
| CN102409088B (en) * | 2011-09-22 | 2014-11-12 | 郭奇伟 | Method for detecting gene copy number variation |
| CN103013989A (en) * | 2011-09-23 | 2013-04-03 | 深圳北京大学香港科技大学医学中心 | Single-nucleotide polymorphisms of cyclin dependent kinase 5 related to senile dementia incidence |
| CN111676281A (en) * | 2019-12-31 | 2020-09-18 | 安徽医科大学第一附属医院 | Kit and method for detecting human mitochondrial ND3 gene 10191 locus genotype based on HRM method |
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Application publication date: 20090909 |