CN103103256B - Can be used for the test kit and the application thereof that detect the DRD4 gene promoter zone methylation degree relevant to schizophrenia - Google Patents
Can be used for the test kit and the application thereof that detect the DRD4 gene promoter zone methylation degree relevant to schizophrenia Download PDFInfo
- Publication number
- CN103103256B CN103103256B CN201210560457.9A CN201210560457A CN103103256B CN 103103256 B CN103103256 B CN 103103256B CN 201210560457 A CN201210560457 A CN 201210560457A CN 103103256 B CN103103256 B CN 103103256B
- Authority
- CN
- China
- Prior art keywords
- methylation
- schizophrenia
- primer
- test kit
- gene promoter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the test kit and application thereof that can be used for detecting the DRD4 gene promoter zone methylation degree relevant to schizophrenia, feature is that this test kit comprises a pair DRD4 gene promoter zone methylation specificity amplification primer and a methylation-specific sequencing primer, wherein upstream primer has the nucleotide sequence as shown in SEQ ID NO.1, downstream primer has the nucleotide sequence as shown in SEQ ID NO.2, methylation-specific sequencing primer is as shown in SEQ ID NO.3, advantage is that this diagnostic kit can realize schizoid detection quickly and easily on a molecular scale, detection efficiency is high, with strong points, simultaneously, the medicine being target spot with DRD4 gene promoter zone methylation is expected to become schizophrenia auxiliary diagnosis, a kind of new tool detecting and screen.
Description
Technical field
The present invention relates to the schizoid detection kit of a kind of auxiliary diagnosis, especially relate to a kind of test kit and the application thereof that can be used for detecting the DRD4 gene methylation degree relevant to schizophrenia.
Background technology
Schizophrenia (Schizophrenia) is one of great mental disorder, and schizophrenia can cause the collapse of form of thinking and emotional reactions, concrete manifestation is felt, consciousness, thinking, the obstacle such as emotion and act of will.Schizophrenia is the disease that a kind of inherited genetic factors and the acting in conjunction of Environmental Psychology factor cause.It is reported, Chinese current schizophreniac is up to 7,800,000., just there is a schizophreniac in the whole world in every 100 people.Although current psychiatry research is mainly devoted to neuroscience field, do not find out rational pathogenesis so far.Therefore, the schizophrenia research carrying out feasibility has very large prospect.
Dopamine Receptors D4(DRD4) be distributed in limbic midbrain area, be by a kind of g protein coupled receptor of genes encoding, and DRD4 gene is positioned at 11p15.5(o.11 the short arm of a chromosome 15 district 5 is with).Current research both domestic and external verified " schizoid clinical symptom due to central dopamine hyperaction cause ".And methylating of gene is not changing in DNA sequence dna situation, affecting genetic expression to have research background to show.Whether there is dependency between gene methylation and schizophrenia to need to be verified further.At present, any correlative study about the test kit for detecting the DRD4 gene methylation degree relevant to schizophrenia report is not also disclosed both at home and abroad.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of test kit and the application thereof that can be used for detecting the DRD4 gene methylation degree relevant to schizophrenia, wherein DRD4 gene methylation level and prevalence of schizophrenia are negative correlation, detection efficiency is high, with strong points.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of test kit that can be used for the detection DRD4 gene methylation degree relevant to schizophrenia, this test kit comprises a pair DRD4 gene promoter zone methylation specificity amplification primer and methylation-specific sequencing primer, wherein
The nucleotide sequence of described methylation-specific amplification upstream primer is as shown in SEQ ID NO.1:
5’-GTGAATTTAGGAGGTTGGGGTAGA-3’；
The nucleotide sequence of described methylation-specific amplification downstream primer is as shown in SEQ ID NO.1:
5’-Biotin-CAAAAAAACAAACAACCCCTCTAA-3’；
Described methylation-specific sequencing primer is as shown in SEQ ID NO.3:
5’-TTGGGGTAGAGATTAGT-3’。
Can be used for an application for the test kit detecting the DRD4 gene methylation degree relevant to schizophrenia, in periphery blood examination is surveyed, this test kit can be used in the schizoid auxiliary diagnosis of males, detection or examination medicine.
Compared with prior art, the invention has the advantages that: the present invention makes public for the first time the test kit and application thereof that can be used for detecting the DRD4 gene methylation degree relevant to schizophrenia, the hyper-methylation level of DRD4 gene promoter region causes the low expression of DRD4 gene, thus affect the internal circulating load that DRD4 transports at brain path and reduce, finally cause schizoid generation and development.Therefore DRD4 gene methylation level and prevalence of schizophrenia are negative correlation, can realize schizoid detection quickly and easily on a molecular scale by the diagnostic kit detected based on DRD4 gene promoter zone methylation level, detection efficiency is high, with strong points, meanwhile, the medicine being target spot with DRD4 gene promoter zone methylation is expected to become a kind of new tool of schizophrenia auxiliary diagnosis, detection and screening.
Accompanying drawing explanation
The association analysis result of 5 CpG points that Fig. 1 is detected sequence region for DRD4 gene and detects; (relation conefficient of such as CpG1 and CpG2 be 0.98, CpG2 and CpG3 relation conefficient be 0.95)
Fig. 2 is methylation level detected result example: represent methylation, and the methylation as illustrated CpG1 to CpG5 is respectively 76%, 68%, 87%, 100%, 98%, 87%; In figure, dash area display is the strength of signal in corresponding CpG site, can analyze obtain its methylation level (upper values that namely dash box is corresponding) in conjunction with this location proximate base kind.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Specific embodiment
1, the collection of research object
Schizophreniac is collected from certain hospital, 354 examples altogether, collect 300 normal persons as a control group simultaneously, through the analysis of age (age of final selected sample is all about thirty years old), sex, knowledge background and these data of DNA related concentrations, filter out the match index (age, educational background, sex) higher experiment sample amount: 30 schizophreniacs (15 male sex+15 women), 30 Normal groups (15 male sex+15 women).
2, the extraction of genomic dna
Application Lab-Aid820 Full automatic instrument for extracting nucleic acid (Chinese Xiamen Zeesan Biotech Co., Ltd., 500ul system) extract the Whole Blood Genomic DNA of sample, the concentration of gained DNA is detected again, for the detection of DRD4 gene promoter area DNA methylation level by nucleic acid-protein determinator.
3, DNA methylation level determination
This research adopts bisulfite pyrosequencing techniques to carry out DNA methylation level detection to 6 of DRD4 gene promoter area CpG sites (as Fig. 1).The ultimate principle of this technology: after the bisulfite process DNA sample in test kit, using polymerase chain reaction (PCR) amplification again, the methylated cytosine(Cyt) of generation (C) base can be made to remain unchanged, and make that methylated C does not occur and be transformed to uridylic (U), then carry out PCR order-checking by sequencing primer, thus obtain which site and there occurs and methylate.This research adopts PyroMark Assay Design software to carry out design of primers, for the pcr amplification primer of testing and sequencing primer as follows:
(1) methylation-specific upstream primer (Forward primer)
5’-GTGAATTTAGGAGGTTGGGGTAGA-3’（SEQ ID NO.1），
(2) methylation-specific downstream primer (Reverse primer)
5’-Biotin-CAAAAAAACAAACAACCCCTCTAA-3’（SEQ ID NO.2）；
(3) methylation-specific sequencing primer (Sequencing primer)
5’-TTGGGGTAGAGATTAGT-3’（SEQ ID NO.3）。
The concrete steps of above-mentioned amplification are:
A. QIAGEN is adopted
bisulf iotate-treated test kit (EpiTech Bisulfite Kits; Qiagen; #59104) bisulfite conversion is carried out to sample DNA.
B. get DNA sample 20ng transformed in steps A and join Pyromark PCR kit (Pyromark PCR Kit; Qiagen; #978703), and add above-mentioned a pair DRD4 gene promoter zone methylation specificity amplification primer, carry out pcr amplification, amplification condition: the first sex change of 95 DEG C of 15min; Then 95 DEG C of 15s, Tm30s, 72 DEG C of 20s, the annealing reaction of totally 50 circulations; Then extension 72 DEG C of 5min.(note: Tm determines according to race PCR gradient temperature in an experiment)
C. the early-stage preparations of Manganic pyrophosphate complex initiation: add annealing buffer (the PyroMark Annealing Buffer that 45 μ l contain 0.3 μM of above-mentioned methylation-specific sequencing primer in PSQ96 plate in advance; Product article No.: Qiagen; #979009); Transfer in an Eppendorf pipe by needing the sepharose 4B total amount (every sample 3 μ l) of the mixing used; Binding buffer liquid (PyroMark Binding Buffer is added in sepharose 4B; Product article No.: Qiagen; #979006), make average each sample about have the volume of 50 μ l, mixture is mixed; Above mixture is added in PCR primer (50 μ l reaction volume), every sample 50 μ l; PCR primer is mixed 10 minutes at normal temperatures, magnetic bead is combined with vitamin H; In vacuum preparation work station, in four sample panel, add the high purity water of 180ml, 70% ethanol, lavation buffer solution (PyroMark Wash Buffer successively; Product article No.: Qiagen; #979008) and denaturation buffer (the PyroMark Denaturation Solution of 120ml; Qiagen; Product article No.: #979007); Open the pump at vacuum preparation work station, vacuum preparation tool is cleaned 30 seconds in high purity water; Then by vacuum preparation tool (PyroMark Vacuum Prep Filter Probes; Product article No.: Qiagen; #979010) move on in PCR plate, capture sepharose 4B (completing this at magnetic bead in PCR primer was in conjunction with latter three minutes to operate); Pick up PCR plate, check whether that most of magnetic bead has all been attracted on vacuum preparation tool; Vacuum preparation tool is put into 70% ethanol 5 seconds; Then 5 seconds are moved on in denaturation buffer; Move on to again in lavation buffer solution and clean 5-10 second; Turn off pump; Vacuum preparation tool is put into the plate containing sequencing primer, shake, release sepharose 4B (sequencing primer also can finally add); Use high purity water cleaning vacuum preparation tool; The PSQ96 plate being placed with sample is put be heated on hot plate 80 DEG C 2 minutes, then cool to room temperature, can carry out Manganic pyrophosphate complex initiation reaction.
D. Manganic pyrophosphate complex initiation: on PyroMark Q24 Manganic pyrophosphate complex initiation instrument, adopts Pyromark Gold Q24 test kit (Pyromark Gold Q24Reagents; Qiagen; #978802) sample in the PSQ96 plate in step C is checked order, then apply PyroMark CpG software and methylation analysis (methylation level detected result example is shown in Fig. 2) is carried out to result.
4, data analysis
This research adopts SPSS16.0 to carry out finishing analysis to data, we find: have 5 site (CpG1 in 6 detected CpG sites, CpG2, CpG3, CpG4 and CpG6) between there is significant correlation (R>0.8, P<0.001, there is statistical significance, see Fig. 1): (during p<0.05, there is statistical significance, lower same), therefore we are CpG1-CpG4, after CpG6 averages, point gender comparison difference of DRD4 gene promoter zone methylation level between case group and control group, result (see table 1) finds CpG5 and CPG1-4, association (p<0.05) is there is with schizophrenia in CPG6 in the male sex.
Compare with other technologies, this test kit of the present invention's design from the index of the methylation of DRD4 gene, accurately can carry out schizoid screening to sample to be measured.To compare the general meter adopted, on qualitative, have more cogency.Meanwhile, also by the change of methylation, the occurring degree of the state of an illness can tentatively be judged.More effectively, easily detection of dynamic is carried out to patient, have accurately and reliably, flexibly fast and the advantage of economy.
Case group and comparing (n=60) between control group after table 1 layering
| Grouping | Case group | Control group | P value |
| Man (30) | |||
| CpG5 methylation (%) | 96.36±7.49 | 62.78±22.33 | 0.022 |
| CpG1-4, CpG6 methylation (%) | 75.83±13.58 | 54.04±18.27 | 0.005 |
| Female (n=30) | |||
| CpG5 methylation (%) | 85.81±16.25 | 81.92±15.54 | 0.496 |
| CpG1-4, CpG6 methylation (%) | 72.74±14.38 | 70.81±12.44 | 0.668 |
Above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited to above-mentioned citing.Those skilled in the art are in essential scope of the present invention, and the change made, remodeling, interpolation or replacement, also should belong to protection scope of the present invention, protection scope of the present invention is as the criterion with claims.
Claims (1)
1. one kind can be used for the test kit detecting the DRD4 gene methylation degree relevant to male schizophrenia, it is characterized in that: this test kit comprises methylation-specific amplification upstream primer and the methylation-specific amplification downstream primer and methylation-specific sequencing primer of DRD4 gene, and the nucleotide sequence of wherein said methylation-specific amplification upstream primer is as shown in SEQ ID NO.1:
5’-GTGAATTTAGGAGGTTGGGGTAGA-3’；
The nucleotide sequence of described methylation-specific amplification downstream primer is as shown in SEQ ID NO.2:
5’-Biotin-CAAAAAAACAAACAACCCCTCTAA-3’；
Described methylation-specific sequencing primer is as shown in SEQ ID NO.3:
5’-TTGGGGTAGAGATTAGT-3’。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210560457.9A CN103103256B (en) | 2012-12-20 | 2012-12-20 | Can be used for the test kit and the application thereof that detect the DRD4 gene promoter zone methylation degree relevant to schizophrenia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210560457.9A CN103103256B (en) | 2012-12-20 | 2012-12-20 | Can be used for the test kit and the application thereof that detect the DRD4 gene promoter zone methylation degree relevant to schizophrenia |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103103256A CN103103256A (en) | 2013-05-15 |
| CN103103256B true CN103103256B (en) | 2015-10-14 |
Family
ID=48311447
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210560457.9A Expired - Fee Related CN103103256B (en) | 2012-12-20 | 2012-12-20 | Can be used for the test kit and the application thereof that detect the DRD4 gene promoter zone methylation degree relevant to schizophrenia |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103103256B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103276095B (en) * | 2013-06-07 | 2014-12-03 | 浙江省医学科学院 | Primer for quantitatively detecting methylation levels of specific sites of human DMBT1 gene through pyrosequencing method |
| GB2533729A (en) * | 2013-08-21 | 2016-06-29 | T Carrell Douglas | Systems and methods for determining impact of age related changes in sperm epigenome on offspring phenotype |
| EP3859015A1 (en) | 2015-08-06 | 2021-08-04 | The University of Utah Research Foundation | Methods of indentifying male fertility status and embryo quality |
| CN112877419A (en) * | 2021-01-20 | 2021-06-01 | 武汉大学 | DNA methylation marker for predicting schizophrenia occurrence risk, screening method and application |
| CN112813155A (en) * | 2021-01-20 | 2021-05-18 | 武汉大学 | DNA methylation marker for predicting therapeutic effect of antipsychotic drug, screening method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008017038A2 (en) * | 2006-08-02 | 2008-02-07 | Mayo Foundation For Medical Education And Research | Methods for selecting medications |
-
2012
- 2012-12-20 CN CN201210560457.9A patent/CN103103256B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008017038A2 (en) * | 2006-08-02 | 2008-02-07 | Mayo Foundation For Medical Education And Research | Methods for selecting medications |
Non-Patent Citations (4)
| Title |
|---|
| A genetic association study of DNA methylation levels in the DRD4 gene region finds associations with nearby SNPs;Docherty S. J. et al.;《Behavioral and Brain Functions》;20120630;第8卷(第31期);第2页右栏第2段 * |
| Accurate quantification of DNA methylation of DRD4 applying capillary gel electrophoresis with LIF detection;Goedecke S. et al.;《ELECTROPHORESIS》;20090430;第30卷(第8期);第2.2节和图2 * |
| Genomic and Epigenomic Instability, Fragile Sites, Schizophrenia and Autism;Smith C. L. et al.;《Current Genomics》;20100930;第11卷(第6期);第454段左栏最后一段 * |
| 多巴胺受体D2型基因启动子区CpG岛甲基化状态与Ⅰ型和Ⅱ型精神分裂症的关联;张海生等;《中国神经精神疾病杂志》;20120731;第38卷(第7期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103103256A (en) | 2013-05-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103103256B (en) | Can be used for the test kit and the application thereof that detect the DRD4 gene promoter zone methylation degree relevant to schizophrenia | |
| Liu et al. | Pediatric acute lymphoblastic leukemia patients exhibit distinctive alterations in the gut microbiota | |
| CN104232781B (en) | TaqMan probe real-time fluorescence PCR (Polymerase Chain Reaction) method for detecting HLA (Human Leukocyte Antigen)-B*5801 alleles | |
| WO2020048518A1 (en) | Group of genes for molecular typing of medulloblastoma and use thereof | |
| CN104328200B (en) | The detection kit of auxiliary diagnosis alzheimer's disease and detection method thereof | |
| CN103602722B (en) | Can be used for the test kit and the application thereof that detect the DRD3 gene methylation degree relevant to schizophrenia | |
| CN103911456B (en) | A kind of detection kit of auxiliary diagnosis alzheimer's disease and detection method thereof | |
| CN108642180A (en) | Detect the method and kit of SDC2 gene methylations | |
| CN110004222A (en) | A kind of multiple gene detection kit and its application method for antipsychotics medication guide | |
| CN107447042B (en) | Molecular marker for diagnosing active tuberculosis diseases and application thereof | |
| CN106048021B (en) | Primer for detecting P53 gene mutation in microcomponent combines and its application | |
| CN106906210A (en) | A kind of fusion primer combination of rapid build amplification sublibrary | |
| CN103160582B (en) | Kit for detecting methylation degree for GCK (glucokinase) gene associated with type 2 diabetes and application thereof | |
| CN104328201B (en) | A kind of detection kit of auxiliary diagnosis alzheimer's disease and detection method thereof | |
| CN103122378B (en) | Kit capable of being used for detecting methylation degree of alpha-adduction protein gene promoter region related to primary hypertension and application thereof | |
| CN103305604B (en) | Kit for detecting CDKN2B gene promoter region methylation degree related to coronary disease and application thereof | |
| JP6164683B2 (en) | Method for analyzing deletion site of spermatogenesis region on Y chromosome and Y chromosome | |
| CN106544428A (en) | Primer and its application needed for detection BDNF gene promoter zone methylation degree | |
| CN109628594B (en) | One kind long-chain non-coding RNA relevant to liver cancer and its application | |
| CN103014165A (en) | Kit capable of being used for detecting methylation degree of PLA2G7 gene promoter region relevant to coronary heart disease and application of kit | |
| CN104561258B (en) | A kind of detection kit for straight colon cancer early diagnosis and application thereof | |
| CN106011253B (en) | Primer for detecting PDGFRA gene mutation in microcomponent combines and its application | |
| CN104726589A (en) | Kit for detecting methylation degree of colorectal cancer related CMTM3 (CKLF-like MARVEL transmembrane domain containing family member 3) gene promoter region and application of kit | |
| CN103966340B (en) | A kind of detection kit for auxiliary diagnosis alzheimer's disease and application thereof | |
| CN104946751B (en) | BRCA1 genes are g.41244291delT mutated and its application in Computer-aided Diagnosis of Breast Cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151014 Termination date: 20181220 |