CN103146822A - PCR (Polymerase Chain Reaction) kit for detecting SNP (Single Nucleotide Polymorphism) relative to female depression - Google Patents
PCR (Polymerase Chain Reaction) kit for detecting SNP (Single Nucleotide Polymorphism) relative to female depression Download PDFInfo
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Abstract
The invention discloses a PCR (Polymerase Chain Reaction) kit for detecting SNP (Single Nucleotide Polymorphism) relative to female depression. The invention further provides a method for detecting a polymorphic locus and the susceptibility of the polymorphic locus with the female depression. A-equipotential locus of a common single nucleotide polymorphism locus rs694066 on a galanin gene at 11q13.3 area of a human body is analyzed by utilizing the nucleotide sequence and the detection method of the polymorphic locus provided by the invention, namely, the situation that whether a T-equipotential locus exists on a DNA (Deoxyribonucleic Acid) complementary chain or not is applied to auxiliary diagnosis on the depression and is used for judging whether an individual has the susceptibility on the depression, thereby being beneficial for disease prevention, early diagnosis and treatment.
Description
Technical field
The present invention relates to the dependency of the polymorphism of a kind of galanin gene monokaryon glycosides pleomorphism site rs694066 and women's associated depression, the present invention also relates to the PCR detection kit of this polymorphic site, and the purposes of this pleomorphism site aspect prevention, auxiliary diagnosis and treatment Depression in women.This invention belongs to biological technical field.
Background technology
Dysthymia disorders is that sickness rate, disabling property are high, the mental disorder that social danger is serious.Large quantity research thinks that the cause of disease of dysthymia disorders is complicated, relates to the many factors such as heredity, biochemistry, psychology and society environment.Studies show that, the lifetime prevalence of unipolar depression is more than 10%, and the women is 2 times of left and right of the male sex.
Single nucleotide polymorphism (SNP) refers on genomic level by the caused DNA sequence polymorphism of the variation of single core thuja acid.SNP can appear at gene control region and coding region, and the coding that the SNP that appears at the coding region may change gene makes that in albumen, certain amino acid changes and affects its function; Appear at the expression of major effect gene of SNP of gene control region.SNP has close dependency as a kind of early stage sudden change of genetic stability with disease.When the patient obviously surpasses non-patient, namely show this SNP and disease association when the frequency of a kind of SNP, by both haplotypes of comparative analysis and research linkage disequilibrium, can be with the Disease-causing gene location of any the unknown in genome.The effect that SNP brings into play in disease gene is located mainly comprises: the one, seek the SNP that causes a disease in the disease locating area, the appearance of this SNP may directly cause on the gene transcription level and the variation on translation skill, namely changed the composition structure of gene expression amount or gene product protein, thereby caused certain disease to occur or make individual to certain special environment susceptible; The 2nd, SNP is as a genetic marker, with disease or phenotype close linkage.
Galanin is one, and 30 polypeptide that amino acid forms are distributed widely in central nervous system by being positioned the galanin genes encoding on karyomit(e) No. 11, by showing multiple physiological effect from different receptors bind.Multinomial document shows, galanin and monoamine transmitters and stress path in close relations, have adjusting maincenter 5-HT energy and the activity of noradrenergic neuron and effect [Mann JJ.The medical management of depression.N Engl J Med2005, the 353:1819-1834 of expression; Fomaro M, Prestia D, Colicchio S, Perugi G.A systematic, updated review on the antidepressant agomelatine focusing on its melatonergic modulation.Curr Neuropharmacol.2010,8 (3): 287-304].Simultaneously, by giving to affect after rat intracerebral ventricle injection galanin or its different receptor stimulant or antagonist behavior depression [the Bing O of rat, Moller C, Engel J.A, et al.Anxiolytic-like action of centrally administered galanin.Neurosci Lett.1993,164 (1-2): 17-20; Bartfai T, Lu X, Badie-Mahdavi H, et al.Galmic, a nonpeptide galanin receptor agonist, affects behaviors in seizure, pain, and forced-swim tests.Proc.Natl.Acad.Sci.USA.2004,101 (28): 10470-10475].And, galanin receptors antagonist M40 can the blocking agent fluoxetine antidepressant effect [Lu X, Barr AM, Kinney JW, et al.A role for galanin in antidepressant actions with a focus on the dorsal raphe nucleus.Proc Natl Acad Sci USA.2005,102 (3): 874-879.].Therefore, the galanin gene may be a tumor susceptibility gene of dysthymia disorders.
by the neurodevelopment experimental rat, studies show that of sheep, the expression of galanin may be subject to estrogenic adjusting, and galanin has the distribution of sex bipolarity at the content of body, be that the jenny in-vivo content is significantly higher than male [Mitchell V, Bouret S, Prevot V, et al.Evidence for expression of galanin receptor Gal-R1mRNA in certain gonadotropin releasing hormone neurones of the rostral preoptic area.Journal of neuroendocrinology.1999, 11:805-812].The people such as Paul GU are by studying also discovery to European patients with depression and anxiety patient, and the serious symptom of galanin gene pleiomorphism and female patient is closely related, does not find this association [43,44] the male patient.This results suggest, galanin may be the especially tumor susceptibility gene of female patient or Depression in women patient's protectiveness factors of patients with depression.But this result awaits further confirmation.
This research is by carrying out 10 SNPs Genotypings detections of galanin gene to 700 routine patients with depression and 673 routine normal healthy controls persons from Beijing and two places, Henan, found that, in whole sample, pleomorphism site rs694066 site is related with the dysthymia disorders height; After the sex layering, only show highly association in women's sample, and the male patient is related without significance.This result is fully pointed out, and the galanin gene may be the tumor susceptibility gene of Depression in women.
Warp is to existing domestic and foreign literature and patent retrieval, so far there are no any galanin gene polymorphism sites rs694066 report relevant to the Depression in women susceptibility.
Summary of the invention
Purpose of the present invention just is to disclose the dependency of galanin gene polymorphism sites and dysthymia disorders, and the purposes aspect the detection dysthymia disorders.
The object of the invention is to disclose the mononucleotide polymorphic site of galanin gene and the susceptibility of Depression in women, and the purposes of this pleomorphism site aspect screening Depression in women Susceptible population.
The present invention passes through the 700 routine patients with depression (male sex's 324 examples, women's 376 examples) and the research of 10 mononucleotide polymorphism sites of galanin gene of 673 routine normal healthy controls persons (male sex's 313 examples, women's 360 examples) find: galanin gene SNP site rs694066 has remarkable related with dysthymia disorders.The rs694066 pleomorphism site genotype GG frequency of patients with depression group galanin gene significantly reduces, and the AG type increases; Patients with depression group rs694066 allelotrope G frequency reduces, and the A gene frequency raises.After the sex layering, 376 routine Depression in women patients and 360 routine women's health collators include analysis in.The result demonstration, the rs694066 pleomorphism site genotype GG frequency that Depression in women patient organizes the galanin gene significantly reduces, and the AG type increases; Patients with depression group rs694066 allelotrope G frequency reduces, and the A gene frequency raises.324 routine male sex's patients with depression and 313 routine men's health collator's analytical resultss are shown, galanin gene polynorphisms site and disease are without significance.
At first the present invention provides a kind of method that detects the single nucleotide polymorphism of galanin gene, comprises the following steps:
(a) determine the Nucleotide of the 91st SNP of sequence shown in the SEQ ID NO:1 of galanin gene fragment;
(b) detecting whether there is mononucleotide polymorphic in described position, namely have A/G (being labeled as r in sequence table) at the 91st, is the T/C loci on its DNA complementary strand.
A second aspect of the present invention is to provide a kind of isolating nucleic acid, has the sequence shown in SEQ ID NO:1, and the 91st is A/G.
The 3rd aspect of the present invention provides a pair of specific nucleic acid primer, have sequence shown in SEQ ID NO:2 or SEQ ID NO:3, and amplify specifically shown in the SEQ ID NO:1 that contains galanin Gene Partial sequence the amplified production of the 91st mononucleotide polymorphic in sequence.
The sequence of preferred nucleic acid primer is as shown in SEQ ID NO:2 or SEQ ID NO:3, and length is respectively 22bp, can specificly amplify the sequence shown in SEQ ID NO:1, and this sequence comprises the rs694066 mononucleotide polymorphic site.
The 4th aspect of the present invention also provides a kind of dysthymia disorders Susceptible population to carry out genetic screening PCR test kit.This test kit detects galanin gene rs694066 loci polymorphism based on allele-specific PCR (allele specific PCR, AS-PCR).This test kit is by the primer that separates packing, without MgCl
2Damping fluid, high-fidelity DNA polymerase, MgCl
2, dNTP material and water forms, wherein the sequence of primer is:
Upstream primer: 5 '-TCATGTCAAATATAGCCGACTT-3 ' is (SEQIDNO:2);
Downstream primer: 5 '-AGAACCGTACACAGATCAAG-3 ' (SEQID NO:3);
rs694066_AF：5′-GTTCTAAGTCCTCTGCCATGCCA-3′(SEQ?ID?NO：4)；
rs694066_AR：5′-GGTCCAGCCTCGTTTTTCCT-3′(SEQ?ID?NO：5)；
rs694066_GF：5′-GTTCTAAGTCCTCTGCCATGCCG-3′(SEQ?ID?NO：6)；
rs694066_GR：5′-ACGGGCAATGAGGTCAAGAG-3′(SEQ?ID?NO：7)。
The concrete grammar in test kit detection rs694066SNP site is as follows:
1. the extraction of sample gene group DNA: the DNA extraction method according to routine is carried out DNA extraction;
2. the pcr amplification of goal gene fragment (comprising sequence shown in SEQ ID NO:1): take sample gene group DNA as template, use, downstream primer amplifying target genes fragment;
3.rs694066 loci polymorphism detects: go on foot the gene fragment of pcr amplification gained as template, respectively with rs694066_A+rs694066_AR and rs694066_G+rs694066_GR primer pair amplification polymorphism locus gene fragment take the 2nd;
4. result is judged: 1.5% agarose gel electrophoresis detects clip size, and the genotype in rs694066 site is A when expanding fragment length is 125bp, and loci is T on its complementary strand; The genotype in rs694066 site is G when expanding fragment length is 283bp, and loci is C on its complementary strand.
Utilize the nucleotide sequence of the polymorphic site relevant to dysthymia disorders provided by the present invention, can build the test kit that dysthymia disorders Susceptible population is carried out the genetics screening.
Test kit of the present invention is easy to use, only needs that amplified production is done the agarose gel electrophoresis detection and gets final product, and need not order-checking, for the low sequenator that need not of the requirement of instrument, detects required time less.
Description of drawings
The somatotype sectional drawing of Fig. 1 Genemapper to pleomorphism site rs694066.
Embodiment
Can more easily understand content of the present invention by consulting following embodiment, these embodiment are just for further illustrating the present invention, and do not mean that restriction scope of the present invention.
The experimental technique that uses in following embodiment is ordinary method if no special instructions.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment one collects blood sample and extracts genomic dna
All research objects come from November, 2008 to 2010 year Anding Hospital, the attached Beijing of the Capital University of Medical Sciences in July, the 6th hospital of Peking University, Beijing Back Long View hospital, Hunan refined the second hospital in Hunan of Central South University,
The outpatient service of lunatic asylum, Henan Province and or inpatient and each pericentral healthy individuals.All experimenters all carry out the SCID-I examination.Enter the group objects age all between 18～60 years old, Han nationality, get rid of great physical disease person.Galanin gene pleiomorphism and dysthymia disorders association study are included patients with depression 700 examples altogether in, the male sex's 324 examples, women's 376 examples).All collators of control group all from each research around the branch center, the same period is healthy, Han nationality is individual, the age, getting rid of had great physical disease and mental disorder, includes 673 routine normal healthy controls persons (male sex's 313 examples, women's 360 examples) between 18～60 years old.This research is through the approval of Anding Hospital, the attached Beijing of Capital University of Medical Sciences ethics comittees.The agreement of research object is all passed through in the Data acquisition, of research object, the collection of blood sample, and the signature Informed Consent Form.All patients extract periphery ulnar vein blood 5mL, are placed in the EDTA anticoagulant tube, and-80 ℃ of preservations are concentrated afterwards and extracted DNA.
The German Macherey-Nagel of DNA extraction employing company DNA extraction test kit.Carry out the DNA extraction step according to the test kit specification sheets as follows: in the whole blood of 200 μ L, add the Proteinase K of 25 μ L, then add 200 μ L B3 solution, concussion 20-30s, 70 ℃ of water-bath 10-15min; The dehydrated alcohol that adds again 210 μ L after mixing, moves in extraction column the centrifugal 1-2min of 11000g/min; Add again 500 μ L BW liquid, the centrifugal 1min of 11000g/min; Add again 600 μ L B5 liquid, the centrifugal 1min of 11000g/min; The seasoning extraction column adds the TE liquid of 70 ℃, standing 1min under room temperature, and the centrifugal 1min of 11000g/min extracts DNA in solution TE ,-80 ℃ of preservations.Utilize ultraviolet spectrophotometer to detect the OD of DNA sample
260And OD
280, 1OD
260Be equivalent to 50ng/ μ L double-stranded DNA.DNA purity is 1.7～2.0.
The acquisition of embodiment two SNP
By the state-run bioinformation of the past document and U.S. retrieval dbSNP (the Datebase of single Nuleotide Polymorphisms of information center, dbSNP) database (http://www.ncbi.nlm.nih.gov/SNP/), and international HapMap database (http://www.hapmap.org/), according to the linkage disequilibrium degree between the SNP site, choose 10 the frequency distribution situations of SNP site in case group and control group at the galanin gene and detect.The PCR reaction is carried out on PERKIN ELMER Gene Amp PCR system9600PCR amplification instrument.SNP detects and carries out on ABI PRISM377DNA sequential detection instrument.
PCR reaction system and program are as follows:
Every duplicate samples is carried out the amplified reaction that a cumulative volume is 20 μ L systems, comprises the genomic dna diluent 50ng of preparation in system, 10 * Taq Buffer2 μ L, 20mmol/L dNTP2 μ L, 100mmol/L MgCl
20.6 μ L, 5U/ μ L Taq archaeal dna polymerase 0.2 μ L, each 5pmol of amplimer adds ddH at last
2It is 20 μ L that O joins cumulative volume.The PCR reaction conditions: 95 ℃ of sex change 15min, 35 circulations, each circulation have 3 temperature, 94 ℃ of 30s, 59 ℃ of 1min, 72 ℃ of 1min extend 7min at 72 ℃ at last.
Amplimer is as shown in the table:
Table 1 each gene polymorphism sites primer sequence and length thereof
Analysis software ABIPR1SM sDS2.1 with the aid of pictures (ABI PRISM377 detector carries).
The correlation analysis of embodiment three galanin gene mononucleotide polypeptide and dysthymia disorders
All data all adopt the SPSS16.0 of spss (Statistical Packages for Social Sciences16.0) to carry out the statistical study of data management and general demography data.Adopt chi square test that the frequency distribution of all SNP loci gene types is analyzed, and carry out respectively Hardy-Weinberg genetic equilibrium check (HWE).The check power in each SNP site is used genetic power calculator (http://statgen.iop.kcl.ac.uk/gpc) computed in software, relative parameters setting is as follows: the rare allele frequency is 0.02, morbidity is 0.10, genotype relative risk Aa=2, AA=2, D '=1, α=0.05, sample genetic statistics effect is about 81%.The Plink statistical software is adopted in galanin allelotrope and depression association analysis.Linkage disequilibrium check between two SNP sites is if D ' value＞0.7 is thought the stronger linkage disequilibrium of existence between two sites; Adopt the Haploview computed in software to go out linkage disequilibrium module (LD block).The haplotype that a plurality of SNP site allelotrope forms builds, and the association analysis of haplotype and disease, and " diallele " pattern is adopted in single haplotype check, and overall haplotype adopts " multiple alleles " pattern.The multiple witness of single SNP site association study statistics adopts Bonferroni to proofread and correct namely: Bonferroni proofreaies and correct P value=α/SNP number of sites.The haplotype analysis result adopts permutation (n=10000) permutation test to proofread and correct.Two-tailed test is all got in all checks, and significance level is got α=0.05.Unit point interaction analysis in Plink software is adopted in interaction analysis between gene polymorphism sites.
Adopt the Hardy-Weinberg balance check, allelotrope and genotype check are carried out in 10 SNPs sites, allelotrope and the genotype in all SNPs site of galanin gene all meet Hardy-Weinberg balance (P＞0.05), see Table 2.
Table 2 galanin gene Chinese Han Population SNP site information and HWE check
HWE：Hardy-Weinberg?equilibrium；MAF：minor?allele?frequency；O(HET)：observed?heterozygosity；E(HET)：predicted?heterozygosity。
This research is carried out gene type to 10 SNP sites on 700 routine patients with depression and 673 routine normal healthy controls persons' galanin gene.Allelotrope and the genotype frequency in all SNPs of patients with depression group and normal healthy controls group site see Table 3.The result of study demonstration, the galanin gene SNP site has remarkable related (seeing Table 3) with dysthymia disorders.The rs694066 pleomorphism site genotype GG frequency of patients with depression group galanin gene significantly reduces, and the AG type increases (χ
2=15.911, p=6.73 * 10
-5); Patients with depression group rs694066 allelotrope G frequency reduces, A gene frequency raise (OR=2.216,95%CI=1.472～3.337, χ
2=15.216, P=9.70 * 10
-5).After proofreading and correct through Bonferroni, the p value is still less than 0.01 (0.0018); After proofreading and correct for test10000 time through permutation, the P value is: 0.010.
After the sex layering, 376 routine Depression in women patients and 360 routine women's health collators include analysis in.Result shows (seeing Table 4), and the rs694066 pleomorphism site genotype GG frequency that Depression in women patient organizes the galanin gene significantly reduces, and the AG type increases (χ
2=12.838, P=0.0003); Patients with depression group rs694066 allelotrope G frequency reduces, A gene frequency raise (OR=2.647,95%CI=1.505～4.656, χ
2=12.243, P=0.0005).324 routine male sex's patients with depression and 313 routine men's health collator's analytical resultss are shown (seeing Table 5), and galanin gene polynorphisms site and disease are without significance.
Embodiment four galanin gene rs694066 site polypeptide detection kit
The 4th aspect of the present invention also provides a kind of dysthymia disorders Susceptible population to carry out genetic screening PCR test kit.This test kit detects galanin gene rs694066 loci polymorphism based on allele-specific PCR (allele specific PCR, AS-PCR).This test kit is by the primer that separates packing, without MgCl
2Damping fluid, high-fidelity DNA polymerase, MgCl
2, dNTP material and water forms, wherein the sequence of primer is:
rs694066_F：5′-TCATGTCAAATATAGCCGACTT-3′(SEQIDNO：2)；
rs694066_R：5′-AGAACCGTACACAGATCAAG-3′(SEQID?NO：3)；
rs694066_AF：5′-GTTCTAAGTCCTCTGCCATGCCA-3′(SEQ?ID?NO：4)：
rs694066_AR：5′-GGTCCAGCCTCGTTTTTCCT-3′(SEQ?ID?NO：5)；
rs694066_GF：5′-GTTCTAAGTCCTCTGCCATGCCG-3′(SEQ?ID?NO：6)；
rs694066_GR：5′-ACGGGCAATGAGGTCAAGAG-3′(SEQ?ID?NO：7)。
The step in test kit detection rs694066SNP site is as follows:
1. the extraction of sample gene group DNA: the DNA extraction method according to routine is carried out DNA extraction;
2. the pcr amplification of goal gene fragment (comprising sequence shown in SEQ ID NO:1): take genomic dna as template, use, downstream primer amplifying target genes fragment.In pcr amplification, the consumption of each component is: concentration is that 50 μ mol upstream primers and concentration are each 0.5 μ mol of downstream primer of 50 μ mol; The damping fluid 2.5 μ L of nothing; Attempt being the archaeal dna polymerase 0.25 μ L of 5U/ μ L; Concentration is the dNTP2 μ L of 2.5mmol; 100mmol/LMgCl
21 μ L; Deionized water 17.75 μ L; Genomic dna sample 1.5 μ L.Goal gene pcr amplification condition: 94 ℃ of denaturation 15min, 94 ℃ of sex change 30s, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, 35 circulations of increasing, 72 ℃ are extended 10min.
3.rs694066 loci polymorphism detects: take above-mentioned 2 described gene fragments as template, respectively with rs694066_A+rs694066_AR and rs694066_G+rs694066_GR primer pair amplification polymorphism locus gene fragment; In pcr amplification, the consumption of each component is: concentration is that 50 μ mol upstream primers and concentration are each 0.5 μ L of downstream primer of 50 μ mol; Without Mg
2+Damping fluid 2.5 μ L; Attempt being the archaeal dna polymerase 0.25 μ L of 5U/ μ L; Concentration is the dNTP2 μ L of 2.5mmol; 100mmol/L MgCl
21 μ L deionized water 17.75 μ L; Template DNA 2 μ L.The pcr amplification condition: 94 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 62 ℃ of annealing 1min, 72 ℃ are extended 1min, 35 circulations of increasing, 72 ℃ are extended 10min.
4. result is judged: 1.5% agarose gel electrophoresis detects clip size, and the genotype in rs694066 site is A when expanding fragment length is 125bp, and loci is T on its complementary strand; The genotype in rs694066 site is G when expanding fragment length is 283bp, and loci is C on its complementary strand.
Illustration with practicality of the present invention
(1) method of setting up can be used for analyzing the A loci that is positioned at the common type mononucleotide polymorphism site rs694066 on the regional galanin gene of 11q13.3 of human body, namely on its DNA complementary strand for the T loci has or not, be applied to the auxiliary diagnosis of dysthymia disorders and the susceptibility whether individuality has a dysthymia disorders passed judgment on.Thereby the prevention, the early diagnosis and therapy that are conducive to disease.
(2) can utilize method and above-mentioned (1) described A loci (namely being the T loci on its DNA complementary strand) with rs694066 that the present invention introduces to carry out the screening of medicine as the medicinal design target spot, seek out to have and regulate the bioactive molecule that galanin is expressed, promote the discovery of new anti-depression drug.
(3) utilize the relevant gene of galanin provided by the invention and the nucleotide sequence of pleomorphism site thereof, build the test kit that dysthymia disorders is carried out genetic diagnosis.
(4) adopt primer sequence provided by the invention can detect special, efficiently galanin gene rs694066 pleomorphism site.
(5) adopt test kit provided by the present invention to detect the polymorphism in Depression in women patient galanin gene rs694066 site, can be used for instructing drug development and clinical rational drug use.
Sequence table
Claims (3)
1.SEQ in the nucleotide sequence of the part galanin gene shown in ID NO:1, the A/G polymorphic sequence of the 91st Nucleotide is in the application for the preparation of the primer in the test kit of Diagnosis of Female dysthymia disorders and/or probe.
2. one kind is carried out genetic screening PCR test kit for Depression in women Susceptible population, it is characterized in that, described test kit comprises: separate the primer of packing, without MgCl
2Damping fluid, high-fidelity DNA polymerase, MgCl
2, dNTP material and water forms; The primer and the nucleotide sequence thereof that wherein separate packing are as follows:
Upstream primer: 5 '-TCATGTCAAATATAGCCGACTT-3 ' is (SEQIDNO:2);
Downstream primer: 5 '-AGAACCGTACACAGATCAAG-3 ' (SEQID NO:3);
rs694066_AF：5′-GTTCTAAGTCCTCTGCCATGCCA-3′(SEQ?ID?NO：4)；
rs694066_AR：5′-GGTCCAGCCTCGTTTTTCCT-3′(SEQ?ID?NO：5)；
rs694066_GF：5′-GTTCTAAGTCCTCTGCCATGCCG-3′(SEQ?ID?NO：6)；
rs694066_GR：5′-ACGGGCAATGAGGTCAAGAG-3′(SEQ?ID?NO：7)。
3. the described test kit of claim 2 in the using method that detects the rs694066 loci polymorphism, comprises the following steps:
1) extraction of sample gene group DNA: the DNA extraction method according to routine is carried out DNA extraction;
2) pcr amplification of goal gene fragment: take the 1st) genomic dna is template the step, use, downstream primer amplifying target genes fragment, the nucleotides sequence of described upstream primer is classified SEQ IDNO:2 as, and the nucleotides sequence of described downstream primer is classified SEQ IDNO:3 as; In pcr amplification, the consumption of each component is: concentration is that 50 μ mol upstream primers and concentration are each 0.5 μ mol of downstream primer of 50 μ mol; The damping fluid 2.5 μ L of nothing; The archaeal dna polymerase 0.25 μ L of 5U/ μ L; Concentration is the dNTP2 μ L of 2.5mmol; 100mmol/L MgCl
21 μ L; Deionized water 17.75 μ L; Genomic dna sample 1.5 μ L.Goal gene pcr amplification condition: 94 ℃ of denaturation 15min, 94 ℃ of sex change 30s, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, 35 circulations of increasing, 72 ℃ are extended 10min.
3) rs694066 loci polymorphism detection: take the 2nd) gene fragment of step gained is template, respectively with rs694066_A+rs694066_AR and rs694066_G+rs694066_GR primer pair amplification polymorphism locus gene fragment; In pcr amplification, the consumption of each component is: concentration is that 50 μ mol upstream primers and concentration are each 0.5 μ L of downstream primer of 50 μ mol; Without Mg
2+Damping fluid 2.5 μ L; Attempt being the archaeal dna polymerase 0.25 μ L of 5U/ μ L; Concentration is the dNTP2 μ L of 2.5mmol; 100mmol/L MgCl
21 μ L deionized water 17.75 μ L; Template DNA 2 μ L.The pcr amplification condition: 94 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 62 ℃ of annealing 1min, 72 ℃ are extended 1min, 35 circulations of increasing, 72 ℃ are extended 10min.
4) result is judged: 1.5% agarose gel electrophoresis detects clip size, and the genotype in rs694066 site is A when expanding fragment length is 125bp, and loci is T on its complementary strand; The genotype in rs694066 site is G when expanding fragment length is 283bp, and loci is C on its complementary strand.
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| CN108410867A (en) * | 2018-02-13 | 2018-08-17 | 广州市番禺区中心医院(广州市番禺区人民医院、广州市番禺区心血管疾病研究所) | A kind of marker of new depression |
| CN108531569A (en) * | 2017-03-03 | 2018-09-14 | 上海伯豪医学检验所有限公司 | The gene marker screened for obsessive-compulsive disorder and schizophrenia, depression and its application |
| CN113337596A (en) * | 2021-06-18 | 2021-09-03 | 同济大学 | Application of reagent for detecting MDLN protein or gene mutation in preparation of kit for diagnosing depression |
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| CN103571849A (en) * | 2012-08-06 | 2014-02-12 | 北京市理化分析测试中心 | Gene, detection method and kit used for detecting depressive disorder |
| CN103571849B (en) * | 2012-08-06 | 2015-05-13 | 北京市理化分析测试中心 | Gene, detection method and kit used for detecting depressive disorder |
| CN108531569A (en) * | 2017-03-03 | 2018-09-14 | 上海伯豪医学检验所有限公司 | The gene marker screened for obsessive-compulsive disorder and schizophrenia, depression and its application |
| CN108531569B (en) * | 2017-03-03 | 2021-05-07 | 上海伯豪医学检验所有限公司 | Gene marker for screening obsessive-compulsive disorder, schizophrenia and depression and application thereof |
| CN108410867A (en) * | 2018-02-13 | 2018-08-17 | 广州市番禺区中心医院(广州市番禺区人民医院、广州市番禺区心血管疾病研究所) | A kind of marker of new depression |
| CN113337596A (en) * | 2021-06-18 | 2021-09-03 | 同济大学 | Application of reagent for detecting MDLN protein or gene mutation in preparation of kit for diagnosing depression |
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