CN101985659A - Kit for testing schizophrenia related gene and preparation method thereof - Google Patents
Kit for testing schizophrenia related gene and preparation method thereof Download PDFInfo
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- CN101985659A CN101985659A CN2010105431227A CN201010543122A CN101985659A CN 101985659 A CN101985659 A CN 101985659A CN 2010105431227 A CN2010105431227 A CN 2010105431227A CN 201010543122 A CN201010543122 A CN 201010543122A CN 101985659 A CN101985659 A CN 101985659A
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Abstract
The invention relates to a kit for testing a schizophrenia related gene and a preparation method thereof in the technical field of molecular biology. The kit comprises a PCR reagent group and a LDR reagent group, wherein the PCR reagent group comprises buffer solution, dNTP mixed solution, Taq DNA polymerase, pure water, and four pairs of amplification primers special for PCR, and the LDR reagent group comprises buffer solution, dNTP mixed solution, Taq DNA ligase, pure water, and four pairs of probes special for LDR. The method comprises the steps of: designing and synthesizing PCR primers by using the primer design software PRIMER3, choosing a TM value thereof about 60 degrees, designing and synthesizing the LDR probe which comprises two 5'end probes and a 3'end probe on each locus, and using TAKARA products or New England Biolabs products as the Taq DNA polymerase and the Taq DNA ligase. The invention can realize rapid, efficient and accurate parting for the specific SNP locus in the schizophrenia related gene.
Description
Technical field
What the present invention relates to is test kit of a kind of field of molecular biotechnology and preparation method thereof, specifically a kind of test kit that detects the schizophrenia associated gene and preparation method thereof.
Background technology
Ligase enzyme can be to being connected with two oligonucleotide fragments of the complete complementary of target sequence, and then form one than long oligonucleotide probe, if two the base of oligonucleotide probe junction and target sequence are not exclusively complementary, ligation just can not take place so, just can not produce long oligonucleotide probe yet.According to this principle, the oligonucleotide probe according to the genotype in SNP site design different lengths can detect the genotype in SNP site.According to the length classification of final product, can determine the genotype in SNP site, ligase enzyme detection reaction that Here it is (Ligase Detection Reaction).
Human genome is the final carrier of genetic information, and the difference basic reason between the human individual also is the difference of genome sequence.Single nucleotide polymorphism (SNP) characterizes the genetic marker commonly used of this species diversity just, and general each thousand base pair just include a SNP on human genome.Proofs such as twins test, schizophrenia are a kind of genetic diseasess of complexity, and heredity grade is up to about 80%.Current genetics studys shows that various genetic diseasess all can be characterized by some SNP site, that is to say, some SNP site and genetic diseases present cognation.The appearance of full genome association study is more sought related site strong technical support is provided.These strong related SNP sites can be used as a kind of sign of ill risk.
To the somatotype in SNP site, method commonly used has the TaqMan probe technique of AB company, sequencing technologies etc. now.The TaqMan probe will be from the customization of AB company, and the cycle is long and price is comparatively expensive.Though the sequencing technologies result is comparatively reliable, to the SNP somatotype of specific site, this method relative cost is higher, and spended time is also long.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of test kit that detects the schizophrenia associated gene and preparation method thereof is provided, the present invention is easy and simple to handle, and is with low cost, develops at schizophrenia.Reliable results, good stability, highly sensitive.
The present invention is achieved by the following technical solutions:
The present invention relates to detect the test kit of schizophrenia associated gene, comprise: PCR reagent set and LDR (ligase enzyme detection reaction) reagent set, two reagent set are packaged in the same box, the PCR reagent set comprises: damping fluid, dNTP mixed solution, Taq archaeal dna polymerase, pure water and four pairs of PCR special-purpose amplification primers; The LDR reagent set comprises: damping fluid, dNTP mixed solution, Taq dna ligase, pure water and four groups of LDR application specific probes.
Described PCR reagent set, buffer concentration is ten times of works better concentration, the concentration of dNTP mixed solution is 10mM, the concentration of Taq archaeal dna polymerase is 2unit/ul, four pairs of pcr amplification primers are the dry powder of 2 OD, during use, and first 8000rpm centrifugal ten minutes, add the pure water dissolving then, can use after the concussion evenly.
Described LDR reagent set, buffer concentration are ten times of works better concentration, and the concentration of Taq archaeal dna polymerase is 40unit/ul, four pairs of pcr amplification primers are the dry powder of 2 OD, during use, and first 8000rpm centrifugal ten minutes, add the pure water dissolving then, can use after mixing.
The invention still further relates to preparation method, may further comprise the steps as the test kit of above-mentioned detection schizophrenia associated gene:
1. the PCR primer design reaches synthetic: the PCR primer adopts primer-design software PRIMER3 to carry out design of primers, and the TM value is selected about 60 degree.
2. the design of LDR probe and synthetic: each site of LDR probe comprises three, two 5 ' end probes and one 3 ' end probe.
A. two 5 ' end probe length differ 2 bases, and this difference is reflected in 5 ' end of this probe
B. last base of 3 ' end of two 5 ' end probes is determined according to the genotype in SNP site
C. two 5 ' end probes are being thought not base of complementary of introducing and target sequence apart from the position of 6 bases of 3 ' end, and purpose is to increase the accuracy that detects
D.5 ' 3 ' the terminal sequence that all adopts polyT of 5 ' of the end probe terminal and 3 ' end probe, purpose are the length that product is reached be beneficial to detection.
E. except that above-mentioned 3 descriptions, other positions of 5 ' end probe and target sequence are complementary fully
F.3 ' end probe and target sequence are complementary fully, and add the phosphoric acid modification at its 5 ' end, and its 3 ' end adds FAM fluorescence and modifies
3. Taq archaeal dna polymerase and Taq dna ligase are selected the product of commercially available TAKARA or the product of New England Biolabs (matched reagent (as damping fluid, dNTP etc.) all select for use commercially available common agents it) for use.
The sequence of the primer of each site correspondence was as follows during described PCR primer design reached and synthesizes:
Described LDR probe, the probe sequence of 5 ' end is complementary fully with target sequence except last base, last base is exactly the position in SNP site to be detected, so this base will be according to the genotype design in this site, the length of this section probe of every kind of genotype correspondence should be different, can realize that purpose is to judge the genotype in SNP site at last by the length of ligation product by adding base at probe 5 ' end.In order to distinguish two kinds of probes more exactly, can introduce artificial mutation in the position about general six bases of a base of distance 3 ' end, make this base and the complementation of target sequence portion, the purpose of doing like this is the sensitivity that increases probe, prevent the generation that mismatches, reduce the possibility of mispairing.The probe of 3 ' end is made up of two portions, and first part is and target dna complementary part to modify but first base of their 5 ' end will add phosphoric acid.The second section of 3 ' end probe sequence is a sequence between additionally adding, and can select the structure of polyT for use, and this sequence end has fluorescence to modify.After design is finished, deliver to corresponding company and synthesize.The present invention has verified that further following sequence is feasible:
Embodiment
Below embodiments of the invention are elaborated: following examples have provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.Among the following embodiment, the experimental technique of unreceipted actual conditions, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1
1. blood sample is collected and the extraction of DNA always
The peripheral blood of gathering the volunteer prepares total DNA.Directly adopt the DNA extraction test kit of Qiagen to carry out the extracting of DNA, method for extracting carries out according to the explanation of test kit.Detect the concentration of each dna sample after the extracting with NanoDrop, after allowing every increment originally is adjusted to 10ng/ul, place-20 degree to preserve standby.
2.PCR reaction
Adopt test kit described in the present invention that the peripheral region in above-mentioned four SNP sites is carried out pcr amplification.Be example explanation testing process below with rs6932590:
(1) the specific PCR primer sequence is as follows:
SEQ?ID?NO.5：5’-CTGGGCGACAGAGAGAGATT-3’
SEQ?ID?NO.6：5’-GGAAGTGAAATCAGTATGTCCAA-3’
The used probe sequence of ligase enzyme detection reaction is as follows:
SEQ?ID?NO.13：5’-TTTTTTTTTTTTTTTTCTGTGCCACCAAAAAAAAAAAAG-3’
SEQ?ID?NO.14：5’-TTTTTTTTTTTTTTTTTTCTGTGCCACCAAAAAAAAAAAAA-3’
SEQ?ID?NO.15：5’-Pi-AAGAAGAATTCCATATCTTGGTGGTTTTTTTTTTTTTTT-FAM-3’
(2) dna fragmentation by pcr amplification target SNP place.Process is summarized as follows: the preparation mixed system comprises 1x PCR damping fluid, the 0.13U archaeal dna polymerase, 0.2 μ M upstream primer (SEQ ID NO.5), 0.2 μ M downstream primer (SEQ ID NO.6), every kind of dNTP of 100 μ M, 2 μ L genomic dnas (10ng/uL).
Operating process is carried out on ice, adds archaeal dna polymerase at last, adds the reagent process and notes changing the rifle head, avoids causing crossed contamination.According to the program in the claim, on the PCR instrument, react.
The PCR response procedures: 94 ℃ 5 minutes, 35 circulations (94 ℃ 30 seconds, 57 ℃ 40 seconds, 72 ℃ 1 minute), 72 degree ten minutes.
Described PCR reaction, purpose is that the LDR for the back provides the template of reaction, and the PCR product should be used further to LDR through purifying earlier, and the system and the program of each reaction are all set forth in claims, no longer repeat at this.
3.PCR product detects
After the PCR reaction is finished, the product of 2ul is mixed with the loading dye of 0.5uL (main component is a tetrabromophenol sulfonphthalein), carry out electrophoresis detection then on 1% sepharose, voltage is arranged on 100V, time 20-30 minute.By the Bio-Rad gel imaging system, use GelDoc 2000 programs and observe the product band then, if produce a unique tangible band, and size conforms to the purpose fragment, illustrates and increases successfully.
4.PCR the purifying of product.
We adopt the method for sodium-acetate-ethanol sedimentation to come purified pcr product.Detailed process is as follows: get the 5uLPCR product, add the sodium-acetate of 1uL3M, the concussion mixing is also centrifugal, and then adds the ethanol of 15uL, the concussion mixing, and centrifugal 30 minutes of 3700rpm during end, takes off inversion, carries out being no more than the most at a high speed the centrifugal of 500rpm.And then add the ethanol of 25uL70%, 3700 centrifugal 15 minutes, during end, repeat above-mentioned inversion centrifugal process.After air-dry, add 10uL deionized water dissolving DNA.
5.LDR reaction
PCR product after the application of purified detects the genotype in each SNP site as the template of ligase enzyme detection reaction.Process is as follows: reaction system: 20uL comprises the damping fluid of 1x dna ligase, 25nM (500fmol) LDR probe, the PCR product of 1000fmol purifying, 8U dna ligase.
Reaction conditions: 95 ℃ 2.0 minutes, 94 degree are 30 seconds then, 65 degree were kept 20 circulations, following 10 minutes of last 95 degree conditions in 1 minute.
Notice that operating process is carried out on ice, adds dna ligase at last, adds the reagent process and notes changing the rifle head, avoids causing crossed contamination.According to the program in the claim, on the PCR instrument, react.
6, the LDR product detects
Can adopt ABI377, perhaps instrument such as ABI3730 detects, and operates according to the working specification of corresponding instrument.Length according to product is judged the range gene type.If produce the band of two kinds of length, be exactly heterozygote so.If have only a kind of band of length, be exactly homozygote so, need judge concrete genotype according to the length contrast of bar interband.
The genotype detection process and the said process in other sites are identical, just adopt the Auele Specific Primer and the probe in other sites.
Present embodiment can carry out somatotype at the MHC zone of schizophrenia association and the genotype in intragenic four the SNP sites of TCF4.Utilize four pleomorphism sites,, can be used for new drug development as the molecular target of medicinal design as one of biomarker.Present embodiment has been set up four sites quick, easy, and detection kit with low cost, this test kit accuracy height, and susceptibility is good, has very strong practical value.
Claims (10)
1. a test kit that detects the schizophrenia associated gene is characterized in that, comprising: PCR reagent set and LDR reagent set, and wherein: the PCR reagent set comprises: damping fluid, dNTP mixed solution, Taq archaeal dna polymerase, pure water, four pairs of PCR special-purpose amplification primers; The LDR reagent set comprises: damping fluid, dNTP mixed solution, Taq dna ligase, pure water, four groups of LDR application specific probes.
2. the test kit of detection schizophrenia associated gene according to claim 1, it is characterized in that, described PCR reagent set, the concentration of dNTP mixed solution is 10mM, and the concentration of Taq archaeal dna polymerase is 2unit/ul, and four pairs of pcr amplification primers are the dry powder of 2 OD, during use, the 8000rpm of elder generation centrifugal ten minutes adds the pure water dissolving then, can use after the concussion evenly.
3. according to the test kit of the detection schizophrenia associated gene described in claims 1, it is characterized in that, described LDR reagent set, the concentration of Taq archaeal dna polymerase is 40unit/ul, four pairs of pcr amplification primers are the dry powder of 2 OD, during use, and first 8000rpm centrifugal ten minutes, add the pure water dissolving then, can use after mixing.
4. the preparation method of the test kit of a detection schizophrenia associated gene according to claim 1 is characterized in that, may further comprise the steps:
1. the PCR primer design reaches synthetic: the PCR primer adopts primer-design software PRIMER3 to carry out design of primers, and the TM value is selected about 60 degree;
2. the design of LDR probe and synthetic: each site of LDR probe comprises three, two 5 ' end probes and one 3 ' end probe;
3. Taq archaeal dna polymerase and Taq dna ligase adopt the product of TAKARA or the product of New England Biolabs.
5. the preparation method of the test kit of detection schizophrenia associated gene according to claim 4 is characterized in that, described LDR probe: two 5 ' end probe length differ 2 bases, are reflected in 5 ' end of this probe.
6. the preparation method of the test kit of detection schizophrenia associated gene according to claim 4 is characterized in that, described LDR probe: last base of 3 ' end of two 5 ' end probes is determined according to the genotype in SNP site.
7. the preparation method of the test kit of detection schizophrenia associated gene according to claim 4, it is characterized in that, described LDR probe: two 5 ' end probes are being thought not base of complementary of introducing and target sequence apart from the position of 6 bases of 3 ' end, purpose is to increase the accuracy that detects.
8. the preparation method of the test kit of detection schizophrenia associated gene according to claim 4, it is characterized in that, described LDR probe: 3 ' the terminal sequence that all adopts polyT of 5 ' terminal and 3 ' end probe of 5 ' end probe, purpose are the length that product is reached be beneficial to detection.
9. the preparation method of the test kit of detection schizophrenia associated gene according to claim 4 is characterized in that, described LDR probe: except that above-mentioned 3 descriptions, other positions of 5 ' end probe and target sequence are complementary fully.
10. the preparation method of the test kit of detection schizophrenia associated gene according to claim 4 is characterized in that, described LDR probe: 3 ' end probe and target sequence are complementary fully, and add the phosphoric acid modification at its 5 ' end, and its 3 ' end adds FAM fluorescence and modifies.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108823303A (en) * | 2018-07-05 | 2018-11-16 | 广州海思医疗科技有限公司 | A kind of detection kit and its purposes for assessing schizophrenia genetic risk |
| CN109549654A (en) * | 2017-09-26 | 2019-04-02 | 上海添音生物科技有限公司 | A kind of common mental disease assisting sifting kit and method |
| CN111334604A (en) * | 2020-04-01 | 2020-06-26 | 上海市农业科学院 | PCR/LDR molecular marker and method for identifying low temperature resistant gene COLD1 genotype of rice |
| CN111909997A (en) * | 2020-09-09 | 2020-11-10 | 上海交通大学 | Application of miRNA marker in preparation of product for evaluating curative effect of olanzapine on schizophrenia treatment and kit |
| CN110964791B (en) * | 2019-12-26 | 2023-08-15 | 贵州中医药大学第二附属医院 | Method for detecting single nucleotide polymorphism and corresponding kit |
-
2010
- 2010-11-13 CN CN2010105431227A patent/CN101985659A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109549654A (en) * | 2017-09-26 | 2019-04-02 | 上海添音生物科技有限公司 | A kind of common mental disease assisting sifting kit and method |
| WO2019063026A1 (en) * | 2017-09-26 | 2019-04-04 | 上海添音生物科技有限公司 | Method and test kit for supplementary screening of common mental disorders |
| CN108823303A (en) * | 2018-07-05 | 2018-11-16 | 广州海思医疗科技有限公司 | A kind of detection kit and its purposes for assessing schizophrenia genetic risk |
| CN110964791B (en) * | 2019-12-26 | 2023-08-15 | 贵州中医药大学第二附属医院 | Method for detecting single nucleotide polymorphism and corresponding kit |
| CN111334604A (en) * | 2020-04-01 | 2020-06-26 | 上海市农业科学院 | PCR/LDR molecular marker and method for identifying low temperature resistant gene COLD1 genotype of rice |
| CN111909997A (en) * | 2020-09-09 | 2020-11-10 | 上海交通大学 | Application of miRNA marker in preparation of product for evaluating curative effect of olanzapine on schizophrenia treatment and kit |
| WO2022052678A1 (en) * | 2020-09-09 | 2022-03-17 | 上海交通大学 | Application of mirna marker in preparing product for therapeutic effect assessment of olanzapine in treating schizophrenia, and test kit |
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Application publication date: 20110316 |