CN111690734B - Primer group for detecting human IFITM5 gene mutation and kit thereof - Google Patents
Primer group for detecting human IFITM5 gene mutation and kit thereof Download PDFInfo
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Abstract
The invention provides a primer group for detecting human IFITM5 gene mutation and a kit thereof, belonging to the technical field of gene detection. The invention relates to a primer group for detecting IFITM5 gene mutation including 5'UTR and 3' UTR of human and a kit thereof, comprising a primer group consisting of PCR amplification primers for detecting IFITM5 gene mutation including 5'UTR and 3' UTR of human; PCR amplification reagents; PCR product purification reagents; a sequencing reagent containing the sequencing primer group for detecting the IFITM5 gene mutation. The invention adopts the specific primer group to detect the gene mutation site aiming at the 5'UTR to 3' UTR region of the IFITM5 gene, and can provide a basis for the gene detection of V-type osteogenesis imperfecta and osteogenesis imperfecta caused by the gene mutation and other potential diseases.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a primer group for detecting human IFITM5 gene mutation and a kit thereof.
Background
Osteogenesis Imperfecta (OI) is a rare disease of inherited connective tissue caused by a collagen anabolic disorder. The main clinical phenotypes included osteoporosis and increased bone fragility, blue sclera, dentinal insufficiency, early-maturing ear sclerosis, and other symptoms. Osteogenesis imperfecta has genetic heterogeneity, and has three genetic modes of autosomal dominant inheritance, autosomal recessive inheritance and X-linked inheritance, wherein more than 80 percent of patients are caused by mutation of collagen type I structural gene COLIA1 or COLlA2, and belong to autosomal dominant inheritance. The V-shaped osteogenesis imperfecta is autosomal dominant inheritance, the main clinical features of patients include repeated fracture, radius head dislocation and hypertrophic callus, and the sclera is generally normal in color. The imaging characteristics include metaphyseal dense shadow, periosteal calcification and the like.
The pathogenic gene of type V osteogenesis imperfecta is IFITM5(interferon induced transmembrane proteins5), which belongs to the interferon-induced transmembrane protein family. The IFITM5 gene is located in chromosome region 11p15.5, and the gene structure includes two exons and one intron. The molecular weight of the encoded protein is 14.8Kda, the encoded protein consists of 132 amino acids, and the structure comprises an extracellular N terminal, a C terminal and an intracellular loop ring. Where the N-and C-termini are the regions most likely to interact with other proteins or cells. The gene domain of IFITM5 is similar to other IFITM family members, but it lacks interferon response elements, and therefore the gene is not inducible by interferon. IFITM5, also known as bone-limiting IFITM-like protein (BRIL), is a bone-specific modulator, and mRNA and protein of IFITM5 are highly expressed on osteoblasts of long bones, vertebrae, facial bones, and periosteum. The expression level of IFITM5 increases with osteoblast differentiation and peaks at the early mineralization and maturation stages of osteoblasts. There were skeletal abnormalities in IFITM5 deficient mice, and the effect on bone formation was more pronounced in neonatal mice than in young and adult mice.
The V-type osteogenesis imperfecta is caused by site-directed mutation of C-14C > T in an upstream 5' -UTR region of an IFITM5 gene, so that after mutation, 5 amino acids are added at the amino terminal of an IFITM5 protein, and the binding of extracellular ligands and receptors is influenced. In addition to site-directed mutations in 5' UTRC. -14C > T causing type V osteogenesis imperfecta, mutations in the coding region of the gene have been reported in patients with non-type V osteogenesis imperfecta, such as mutations at the c.119C > T (p.Ser40Leu) site, and corresponding patients develop skeletal lesions that do not conform to typical type V osteogenesis imperfecta, but resemble type VI limb shortening. Patients with the c.119c > G (p.ser40trp) mutation had mild clinical symptoms and the first fracture occurred at 11 years of age.
The next-generation sequencing technology (next-generation sequencing) plays an important role in the discovery of pathogenic genes of genetic diseases and the like, and particularly plays an important role in the mutation detection of unknown pathogenic genes and structurally complex genes. The next generation sequencing technology has the characteristics of high flux, large data volume, low detection cost, parallel analysis and the like, but the operation flow is complex, the high-flux sequencing data needs to be subjected to complex bioinformatics analysis, the specificity is strong, and the obtained mutation sites need Sanger sequencing verification.
Sanger sequencing, as a traditional one-generation sequencing technology, has been the gold standard for gene detection. The DNA molecule is directly sequenced, the operation process is simple and easy to implement, the speed is high, the accuracy is high, the standardization can be realized, the sequencing result is visual, and the analysis is convenient. The kit is suitable for detecting the pathogenic genes of the genetic diseases of single genes or multiple genes with limited quantity, known pathogenic genes, known mutation sites and simple structures. The method has no advantages for detection of a large number of candidate genes and a large number of samples without defining the candidate genes. Meanwhile, for Sanger sequencing, sequencing fragments are short, and mutation types such as large fragment deletion, copy number variation and the like cannot be detected, so that detection results are missed.
Disclosure of Invention
In view of this, the present invention aims to provide a primer set for detecting human IFITM5 gene mutation and a kit thereof, wherein the primer set enables the determination result to have higher accuracy and specificity.
The invention provides a primer group for amplifying a human IFITM5 gene containing a 5'UTR and a 3' UTR, which comprises 3 pairs of primers;
the primers for PCR amplification from 5' UTR of IFITM5 gene to exon 1 are upstream primer FP1 with nucleotide sequence shown as SEQ ID NO.1 and downstream primer RP1 with nucleotide sequence shown as SEQ ID NO. 2;
the primers for PCR amplification of the exon 2 of the IFITM5 gene are an upstream primer FP2 with the nucleotide sequence shown as SEQ ID NO.3 and a downstream primer RP2 with the nucleotide sequence shown as SEQ ID NO. 4;
the primers for PCR amplification of the 3' UTR of the IFITM5 gene are an upstream primer FP3 with the nucleotide sequence shown as SEQ ID NO.5 and a downstream primer RP3 with the nucleotide sequence shown as SEQ ID NO. 6.
The invention provides a primer group for detecting human IFITM5 gene mutation containing 5'UTR and 3' UTR, which comprises 3 primers for sequencing exons of IFITM5 gene from 5'UTR to 3' UTR;
the nucleotide sequence of a primer FP1 for PCR sequencing from 5' UTR to exon 1 is shown as SEQ ID NO. 1;
the nucleotide sequence of the PCR sequencing primer FP2 of the exon 2 is shown as SEQ ID NO. 3;
the nucleotide sequence of a PCR sequencing primer RP3 of the 3' UTR is shown in SEQ ID NO. 6.
The invention provides a kit for detecting human IFITM5 gene mutation including 5'UTR and 3' UTR, which comprises a primer group for detecting human IFITM5 gene mutation including 5'UTR and 3' UTR.
Preferably, further comprising DNA sequencing reagents;
the DNA sequencing reagent comprises BigDye3.1 mixed solution, EDTA solution, absolute ethyl alcohol, 75% ethanol water solution with volume concentration and Hi-Di formamide solution.
Preferably, the primer set for amplifying the human IFITM5 gene comprising a 5'UTR and a 3' UTR is also included.
Preference is given toThe kit also comprises PCR amplification reagents; the PCR amplification reagent comprises 10mM dNTP Mix and Mg2+2X Phanta Max Buffer and DNA polymerase.
Preferably, the PCR product purification reagent comprises: SAP enzymes and ExoI enzymes.
The invention provides application of the primer group in preparation of a kit for detecting human IFITM5 gene mutation including 5'UTR and 3' UTR.
The primer group for amplifying the human IFITM5 gene containing 5'UTR and 3' UTR realizes specific amplification of the whole sequence of the human IFITM5 gene containing 5'UTR and 3' UTR, and provides a material for detecting the gene mutation site of the whole sequence of the human IFITM5 gene.
The primer group for detecting the mutation of the human IFITM5 gene including 5'UTR and 3' UTR provided by the invention is used for carrying out mutation detection on IFITM5 gene DNA including 5'UTR and 3' UTR, so that the accuracy and specificity of a measurement result are ensured. Compared with the traditional detection method by adopting a probe method through fluorescent quantitative PCR, the detection cost is reduced, and meanwhile, the accuracy of the PCR product is higher than that of the fluorescent quantitative PCR because the PCR product is completed through DNA sequence determination and subsequent sequence comparison. Compared with high-throughput sequencing, the method directly detects the IFITM5 pathogenic gene, and avoids the problems of false positive and false negative possibly caused by the problems of original data screening, threshold setting and the like in high-throughput sequencing; meanwhile, the cost and the labor are saved.
Meanwhile, the PCR reaction program of each pair of amplification primers provided by the invention is completely consistent with the reaction program of the DNA sequencing primer, so that the amplification sequencing can be carried out simultaneously, and the workload of the detection process is greatly reduced.
Drawings
FIG. 1 is a peak diagram of IFITM5 gene sequencing of 6 cases of patients with osteogenesis imperfecta P1-P6 blood samples in the example of the present invention.
Detailed Description
The invention provides a primer group for amplifying a human IFITM5 gene containing a 5'UTR and a 3' UTR, which comprises 3 pairs of primers; the primers for PCR amplification from 5' UTR of IFITM5 gene to exon 1 are upstream primer FP1 with nucleotide sequence shown as SEQ ID NO.1 and downstream primer RP1 with nucleotide sequence shown as SEQ ID NO. 2; the primers for PCR amplification of the exon 2 of the IFITM5 gene are an upstream primer FP2 with the nucleotide sequence shown as SEQ ID NO.3 and a downstream primer RP2 with the nucleotide sequence shown as SEQ ID NO. 4; the primers for PCR amplification of the 3' UTR of the IFITM5 gene are an upstream primer FP3 with the nucleotide sequence shown as SEQ ID NO.5 and a downstream primer RP3 with the nucleotide sequence shown as SEQ ID NO. 6.
In the invention, the primer group for amplifying the human IFITM5 gene comprising 5'UTR and 3' UTR is applied to the preparation of a kit for amplifying the complete sequence of the human IFITM5 gene. The kit preferably comprises PCR amplification reagents and PCR product purification reagents. The PCR amplification reagent comprises 10mM dNTP Mix and Mg2+2X Phanta Max Buffer and DNA polymerase. The PCR product purification reagent preferably comprises: SAP enzymes and ExoI enzymes.
In the present invention, the method for using the kit preferably comprises the following steps:
1) carrying out PCR amplification on a DNA fragment of the human IFITM5 gene containing a 5'UTR and a 3' UTR by adopting the primer group to obtain a PCR amplification product;
2) and purifying the PCR amplification product to obtain a purified PCR amplification product.
In the present invention, the reaction system for PCR amplification is 40 μ l, and comprises: 10mM dNTP Mix 1. mu.l, 2 XPanta Max Buffer 20. mu.l, DNA polymerase 1. mu.l, upstream primer 4. mu.l, downstream primer 4. mu.l, DNA template 50ng-400ng, made up to 40. mu.l with DEPC water. The upstream primer and the downstream primer are preferably a pair of primers, and the PCR amplification reaction of each pair of primers is separately performed. The reaction procedure for the PCR amplification is preferably as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15 seconds, annealing at 65 ℃ for 15 seconds, extension at 72 ℃ for 30 seconds, and cycle number 35; final extension at 72 ℃ for 5 min.
In the present invention, the purified reaction system is preferably 20. mu.l, comprising: PCR products 8. mu.l, 1U/. mu.l SAP enzyme 1. mu.l, 10U/. mu.l ExoI enzyme 1. mu.l and H2O10μl。
The reaction conditions for the purification are as follows: digestion was carried out at 37 ℃ for 60 minutes and then inactivation was carried out at 65 ℃ for 15 minutes. And (3) purifying the product, performing 1% agarose gel electrophoresis or concentration determination by a trace nucleic acid quantitative analyzer, performing subsequent sequencing, and detecting the gene mutation site.
The invention provides a primer group for detecting human IFITM5 gene mutation containing 5'UTR and 3' UTR, which comprises 3 primers for sequencing exons of IFITM5 gene from 5'UTR to 3' UTR;
the nucleotide sequence of a primer FP1 for PCR sequencing from 5' UTR to exon 1 is shown as SEQ ID NO. 1;
the nucleotide sequence of the PCR sequencing primer FP2 of the exon 2 is shown as SEQ ID NO. 3;
the nucleotide sequence of a PCR sequencing primer RP3 of the 3' UTR is shown in SEQ ID NO. 6.
The invention provides a kit for detecting human IFITM5 gene mutation including 5'UTR and 3' UTR, which comprises a primer group for detecting human IFITM5 gene mutation including 5'UTR and 3' UTR.
In the present invention, each DNA sequencing reaction system is 10. mu.l, and comprises 4. mu.l BigDye, 3.2pMol/L upstream primer 1. mu.l, 3.2pMol/L downstream primer, and 5-20 ng of purified PCR product, and is made up to 10. mu.l with DEPC water. The upstream primer and the downstream primer are preferably a pair of primers, and the PCR amplification reaction of each pair of primers is separately performed. The reaction procedure for the primer set was as follows: denaturation at 96 ℃ for 1 min, denaturation at 96 ℃ for 10 sec, 50 ℃ for 5 sec, and denaturation at 60 ℃ for 4 min for 28 cycles, PCR cycling and cooling to 4 ℃ to remove and immediately centrifuge for a short time.
In the present invention, the kit preferably further comprises a DNA sequencing reagent; the DNA sequencing reagent comprises BigDye3.1 mixed solution, 0.125M EDTA solution, absolute ethyl alcohol, 75% ethanol water solution with volume concentration and Hi-Di formamide solution. The kit preferably further comprises a primer set for amplifying the human IFITM5 gene comprising a 5'UTR and a 3' UTR. The kit preferably further comprises PCR amplification reagents; the PCR amplification reagent comprises 10mM dNTP Mix and Mg2+2X Phanta Max Buffer and DNA polymerase. The PCR product purification reagent preferably comprises 1U/. mu.l SAP enzyme and 10U/. mu.l ExoI enzyme. The source of the above-mentioned reagents is not particularly limited in the present invention, and any commercially available source known in the art may be used.
The invention provides application of the primer group in preparation of a kit for detecting human IFITM5 gene mutation including 5'UTR and 3' UTR.
In the present invention, the components of the kit are preferably the same as those of the kit, and are not described herein again.
The IFITM5 gene mutation group and the detection kit thereof provided by the invention can be used for site-specific monitoring of V-type osteogenesis imperfecta and can also be used for mutation detection of non-V-type osteogenesis imperfecta and other potential diseases caused by mutation of other sites of IFITM5 gene, thereby assisting in clinical diagnosis of diseases according to mutation sites and clinical phenotypes. Meanwhile, a foundation is laid for researching the relationship between IFITM5 gene mutation and gene function and the correlation between genotype and phenotype.
The primer set and the kit for detecting human IFITM5 gene mutation provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Description of the sources of reagents
The whole blood genomic DNA extraction kit was purchased from Omega;
10mM dNTP Mix, Mg-containing2+2X Phanta Max Buffer, Phanta Max Super-Fidelity DNA Polymerase of (g) were from Vazyme;
1U/. mu.l SAP and 10U/. mu.l ExoI were purchased from NEB;
BigDye3.1 sequencing solution, Na2EDTA﹒2H2Both O and Hi-Di formamide are available from ThermoFisher Scientific;
absolute ethanol was purchased from the national pharmaceutical group;
RNase and DNase removed water (DEPC water) was purchased from Life Technologies;
agarose was imported from Spain, and primers were synthesized from the Huada gene.
Example 1
IFITM5 gene DNA sequence (NG _032892.1) was analyzed based on NCBI GeneBank database to design primers for exon amplification from 5'UTR to 3' UTR. The amplification region comprises all exons of IFITM5 and intron sequences of which the boundaries of the exons and the introns are not less than 50 bp. The nucleotide sequences of the primers used in the PCR amplification are shown in Table 1.
TABLE 1 IFITM5 Gene exon primer amplification and sequencing sequences
Example 2
A kit for detecting mutations in the IFITM5 gene, including both 5'UTR and 3' UTR, in a human, comprising:
the primer group consisting of the PCR primers for detecting IFITM5 gene mutation including 5'UTR and 3' UTR;
the PCR amplification reagent comprises: 10mM dNTP Mix, Mg2+2X Phanta Max Buffer, Phanta Max Super-Fidelity DNA Polymerase, RNase and DNase water (DEPC water);
the working concentration of each component in the PCR amplification system is as follows: 10000. mu.M dNTP Mix, 2mM 2X Phanta Max Buffer, 1U/. mu.l Phanta Max Super-Fidelity DNA Polymerase, 5. mu.M upstream primer and 5. mu.l downstream primer, and 50-400 ng/. mu.l template concentration.
The PCR product purification reagent comprises: 1U/. mu.l SAP enzyme, 10U/. mu.l ExoI enzyme, DEPC water; the working concentration of each component in the PCR product purification system is as follows: 0.05U/. mu.l SAP enzyme, 0.5U/. mu.l ExoI enzyme.
The DNA sequencing reagent comprises: the sequencing primer group for detecting IFITM5 gene mutation, BigDye3.1 mixed solution, 0.125M EDTA solution, absolute ethyl alcohol, 75% ethanol water solution by volume percentage, Hi-Di formamide solution, RNase removal and DNase water;
the working concentration of the sequencing primer in the sequencing primer set was 0.64 pmol/L.
The kit is stored at the temperature of-20 ℃, and repeated freeze thawing is reduced as much as possible.
Example 3
The method for detecting the IFITM5 gene mutation by using the kit for detecting the IFITM5 gene mutation including 5'UTR and 3' UTR comprises the following steps:
(1) extraction of peripheral blood genomic DNA
Collecting 5ml of peripheral blood of a patient, extracting whole blood genome DNA by using an Omega DNA extraction kit (D3392-01), and measuring the DNA concentration and purity by using a NanoDrop2000/2000c spectrophotometer; the specific operating step conditions refer to the product specification;
(2) PCR amplification
The PCR reaction system is 40 μ l, which comprises:
10mM dNTP Mix 1. mu.l, 2X Phanta Max Buffer 20. mu.l, Phanta Max Super-Fidelity DNA Polymerase 1. mu.l, upstream primer 4. mu.l, downstream primer 4. mu.l, DNA template 50-400 ng, and made up to 40. mu.l with DEPC water.
Touchdown PCR was carried out using a BIO-RAD PIC-200 PCR apparatus under the following conditions:
pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15 seconds, annealing at 65 ℃ for 15 seconds, extension at 72 ℃ for 30 seconds, and cycle number 35; final extension at 72 ℃ for 5 min.
(3) PCR product purification
The PCR purification system is 20 μ l, which comprises: PCR product 8. mu.l, 1U/. mu.l SAP enzyme 1. mu.l, 10U/. mu.l ExoI enzyme 1. mu.l, H2O10 μ l. The purification reaction conditions are as follows: digestion was carried out at 37 ℃ for 60 minutes and then inactivation was carried out at 65 ℃ for 15 minutes. After purifying the product, the concentration of the product is determined by 1 percent agarose gel electrophoresis or a trace nucleic acid quantitative analyzer.
(4) DNA sequencing reaction and purification
Each DNA sequencing reaction system is 10 μ L, including 4 μ L BigDye, 3.2pMol/L corresponding sequencing primer 2 μ L, 5-20 ng purified PCR product, and is made up to 10 μ L with DEPC water.
The sequencing reaction conditions are as follows: denaturation at 96 ℃ for 1 min, denaturation at 96 ℃ for 10 sec, 50 ℃ for 5 sec, and denaturation at 60 ℃ for 4 min for 28 cycles, PCR cycling and cooling to 4 ℃ to remove and immediately centrifuge for a short time.
After the completion of the sequencing reaction, 2.5. mu.l of 0.125M EDTA solution was added to the reaction product, and then 30. mu.l of anhydrous ethanol was added thereto, and the mixture was inverted and mixed, and then allowed to stand at room temperature for 15 minutes. After centrifugation at 12000g for 10 min, the supernatant was removed. 60. mu.l of 70% aqueous ethanol was added, and after centrifugation at 12000g at 4 ℃ for 15 minutes, the supernatant was removed. Repeating the steps once, dissolving the precipitate into 10 mu lHi-Di formamide after ethanol is completely volatilized, and performing sequencing analysis by using an ABI3130XL sequencer after the precipitate is denatured for 4 minutes at 95 ℃.
(5) Analysis of sequencing results
The ExAC (the Exome Aggregation Consortium) database contains more than 6 ten thousand of personal genome information and gene variation data. The Polyphen2, SIFT and Mutation taster can predict the influence of gene variation on the structure and function of the encoded protein, and evaluate the possible pathogenicity. The Mutation site is identified by using Mutation surface software, the known Mutation frequency of the Mutation site is analyzed by using an ExAC database, and the Mutation function is predicted by using PolyPhen2, SIFT and Mutation pointer.
Experimental example 4
A total of 10 patients clinically diagnosed as osteogenesis imperfecta from the civil Hospital of Wuqing district, Tianjin were subjected to IFITM5 gene testing using the kit described in example 2 and the method described in example 3.
The sequencing peak of the IFITM5 gene from the patient blood sample is shown in FIG. 1. Among them, IFITM5c. -14C > T site-directed heterozygous mutation 9 cases all had the characteristics of radius head dislocation and huge callus formation after fracture. In the case of 1 c.343G > A (p.Ala115Thr), the patients frequently fractured and had decreased bone density and severe skeletal malformation manifestations.
All detected c.343G > A (p.Ala115Thr) variation sites were subjected to mutation effect prediction. Polyphen2 predicts 0.997, SIFT 0.014, and Mutation taster 0.999769673099611. In the prediction, the specific value represents the degree of damage, and the closer the score is to 1, the greater the damage is likely to be. In SIFT prediction, it is considered that the function of the encoded protein may be affected as long as the score is less than 0.05. In the Mutation taster software, scores are all close to 1, which indicates that the influence of the site on the function is all disease-causing. Whereas mutations occurring in conserved regions are generally considered markers of pathogenicity. According to three different functional effect prediction software, the base substitution of the site can influence the function. The IFITM5c.343G > A (p.Ala115Thr) site mutation is a new mutation of the IFITM5 gene and can be inferred to cause osteogenesis imperfecta by combining clinical phenotypes such as abnormal skeletons of patients.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (4)
1. A kit for detecting human IFITM5 gene mutation including 5'UTR and 3' UTR is characterized by comprising a primer set for detecting human IFITM5 gene mutation including 5'UTR and 3' UTR and a primer set for amplifying human IFITM5 gene including 5'UTR and 3' UTR;
the primer group for detecting the mutation of the human IFITM5 gene comprising 5'UTR and 3' UTR comprises 3 primers for sequencing exons of the IFITM5 gene from 5'UTR to 3' UTR; the nucleotide sequence of a primer FP1 for PCR sequencing from 5' UTR to exon 1 is shown as SEQ ID NO. 1; the nucleotide sequence of the PCR sequencing primer FP2 of the exon 2 is shown as SEQ ID NO. 3; the nucleotide sequence of a PCR sequencing primer RP3 of the 3' UTR is shown as SEQ ID NO. 6;
the primer group for amplifying the human IFITM5 gene comprising 5'UTR and 3' UTR comprises 3 pairs of primers;
the primers for PCR amplification from 5' UTR of IFITM5 gene to exon 1 are an upstream primer FP1 with the nucleotide sequence shown as SEQ ID NO.1 and a downstream primer RP1 with the nucleotide sequence shown as SEQ ID NO. 2; the primers for PCR amplification of exon 2 of IFITM5 gene are upstream primer FP2 with the nucleotide sequence shown as SEQ ID NO.3 and downstream primer RP2 with the nucleotide sequence shown as SEQ ID NO. 4; the primer for PCR amplification of the 3' UTR of the IFITM5 gene is an upstream primer FP3 with the nucleotide sequence shown as SEQ ID NO.5 and a downstream primer RP3 with the nucleotide sequence shown as SEQ ID NO. 6.
2. The kit of claim 1, further comprising DNA sequencing reagents; the DNA sequencing reagent comprises BigDye3.1 mixed solution, EDTA solution, absolute ethyl alcohol, 75% ethanol water solution with volume concentration and Hi-Di formamide.
3. The kit of claim 1, further comprising PCR amplification reagents; the PCR amplification reagent comprises dNTP Mix and Mg2+2X Phanta Max Buffer and DNA polymerase.
4. The kit according to any one of claims 1 to 3, further comprising a PCR product purification reagent; the PCR product purification reagent comprises:SAPenzymes andExoi enzyme.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586389A (en) * | 2014-10-21 | 2016-05-18 | 天津华大基因科技有限公司 | Kit and application thereof in detection on hereditary bone disease genes |
| CN109897894A (en) * | 2018-12-27 | 2019-06-18 | 黄欢 | A kind of pathogenic mutation and its detection reagent of the infull disease of Osteogenic developmental |
| CN110358823A (en) * | 2019-07-25 | 2019-10-22 | 内蒙古自治区人民医院 | A kind of primer sets for detecting SFTPB gene mutation, kit and its amplification method |
| WO2020006132A1 (en) * | 2018-06-27 | 2020-01-02 | Altius Institute For Biomedical Sciences | Gapped and tunable repeat units for use in genome editing and gene regulation compositions |
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| CN106755395B (en) * | 2016-12-16 | 2020-05-12 | 山东第一医科大学(山东省医学科学院) | Mutation site of XI type osteogenesis imperfecta pathogenic gene FKBP10 and application thereof |
| CN110684765A (en) * | 2019-09-20 | 2020-01-14 | 浙江大学 | Screening method of dementia-related pathogenic gene capture probe, captured genome, product and application |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586389A (en) * | 2014-10-21 | 2016-05-18 | 天津华大基因科技有限公司 | Kit and application thereof in detection on hereditary bone disease genes |
| WO2020006132A1 (en) * | 2018-06-27 | 2020-01-02 | Altius Institute For Biomedical Sciences | Gapped and tunable repeat units for use in genome editing and gene regulation compositions |
| CN109897894A (en) * | 2018-12-27 | 2019-06-18 | 黄欢 | A kind of pathogenic mutation and its detection reagent of the infull disease of Osteogenic developmental |
| CN110358823A (en) * | 2019-07-25 | 2019-10-22 | 内蒙古自治区人民医院 | A kind of primer sets for detecting SFTPB gene mutation, kit and its amplification method |
Non-Patent Citations (6)
| Title |
|---|
| A Single Recurrent Mutation in the 50-UTR of IFITM5 Causes Osteogenesis Imperfecta Type V;Tae-Joon Cho等;《The American Journal of Human Genetics》;20120810;第91卷;第344页左栏第3段，Table S4 * |
| Gene mutation spectrum and genotype-phenotype correlation in a cohort of Chinese osteogenesis imperfecta patients revealed by targeted next generation sequencing;Y. Liu等;《Osteoporos Int》;20171028;第28卷(第10期);第2985–2995页 * |
| Homo sapiens interferon induced transmembrane protein 5 (IFITM5)，RefSeqGene on chromosome 11;NCBI;《GenBank》;20190825;"ORIGIN" * |
| 大样本成骨不全症基因型—表型关系及唑来膦酸对成骨不全症患儿椎体形态影响;李路娇;《中国博士学位论文全文数据库医药卫生科技辑（月刊）》;20200215(第02期);E066-105 * |
| 成骨不全V型4家系IFITM5基因突变鉴定;张增等;《中华医学会第五次中青年骨质疏松和骨矿盐疾病学术会议论文集》;20130627;第32-33页 * |
| 成骨不全致病基因突变及I型胶原蛋白基因甲基化分析;石贤龙;《中国优秀硕士学位论文全文数据库医药卫生科技辑（月刊）》;20160815(第08期);E066-193 * |
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