CN105695606B - Screening method for hypertrophic cardiomyopathy related pathogenic gene mutation for non-therapeutic purpose - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to a screening method for the mutation of pathogenic genes related to hypertrophic cardiomyopathy for non-treatment purposes, which comprises the following steps: (1) extracting a genome; (2) amplifying the target gene by adopting multiplex PCR; (3) constructing a target gene library; (4) sequencing the target gene library through a next generation semiconductor sequencing platform, and screening out gene mutation sites related to the incidence of hypertrophic cardiomyopathy. The method is simple, convenient and quick, has low cost, can detect a plurality of samples at one time, lays a foundation for screening hypertrophic cardiomyopathy and develops a road for clinical molecular diagnosis.
Description
Technical Field
The invention relates to the field of biotechnology, medical molecular diagnosis and biotechnology, in particular to a screening method for detecting the mutation of a pathogenic gene related to hypertrophic cardiomyopathy for non-treatment purposes.
Background
Hypertrophic Cardiomyopathy (HCM) is one of the most common inherited heart diseases, primarily manifested as left ventricular asymmetric hypertrophy or thickening of the myocardial wall, with fewer patients exhibiting left ventricular outflow obstruction, right ventricular hypertrophy, or biventricular hypertrophy. The pathological features are diffuse hypertrophy, deformity, large nucleus, deep dyeing, myocardial fiber disorder and the like of myocardial cells. HCM is clinically manifested from no signs to dyspnea, syncope, chest pain and even sudden cardiac death and fatal arrhythmias.
HCM is the first inherited cardiac disease that elucidates the cause genetically. The first causative gene, MYH7, was found in 1990 in a canadian patient with familial hypertrophic cardiomyopathy, following an autosomal dominant inheritance pattern. The global morbidity and annual mortality for HCM is approximately 1/500 and 1%, respectively. Relevant evidence shows that Zou et al randomly spot-check 8080 adult people in 9 provinces of China, wherein 4064 male people and 4016 female people, and perform echocardiogram detection on the 8080 adult people, and the results show that the probability of suffering from HCM of Chinese people is about 0.8/500. In fact, echocardiography for hypertrophic cardiomyopathy is a diagnostic method after illness, and there are many more potential HCM patients, so that it is concluded that there are at least 200 million HCM patients in the chinese population. The disease is also one of the leading causes of sudden death in teenagers and young athletes, and statistical analysis of 1886 American young athletes dying suddenly in 1986-.
Related studies have shown that HCM is mainly caused by mutations in sarcomere genes, and HCM is also known as sarcomere disease. HCM mainly follows an autosomal dominant inheritance pattern, with less autosomal recessive inheritance, and about 50% of HCM patients are familial inherited, known as Familial Hypertrophic Cardiomyopathy (FHCM). HCM has a high degree of clinical genetic heterogeneity, and up to now, about 30 pathogenic genes have been found to be associated with HCM pathogenesis, with more than 1400 pathogenic mutation sites, but about 80% of HCM patients have pathogenic genes mainly occurring in genes encoding thick, thin or energy-metabolizing proteins, because: MYH7, MYBPC3, TNNT2, TNNI3, MYH6, TPM1, ACTC1, PRKAG2, MYL2, and MYL 3. The mechanism by which sarcomere mutations lead to HCM is not well understood, and it is presently believed that sarcomere gene mutations result in Ca pairs in thick or thin filaments2+The affinity and the sensitivity of the myocardial cells are enhanced, the energy in the cells is excessively consumed, the energy supply of the myocardial cells is insufficient, the death of the myocardial cells is triggered, and the electric conduction capability of the myocardial cells is impairedAnd thus, ventricular hypertrophy.
The gene detection and the family screening of the hypertrophic cardiomyopathy patient can provide important guidance for clinical diagnosis, and mainly comprise the following steps: 1) prenatal diagnosis, guiding prepotency; 2) assisting in definite diagnosis and carrying out clinical intervention; 3) and (4) family screening, and performing family disease occurrence risk assessment and management.
The hypertrophic cardiomyopathy is characterized by clinical genetic heterogeneity and more pathogenic genes, and the number of pathogenic mutation sites is close to 1400. CN 102965428A discloses a preparation kit for detecting genetic cardiac hypertrophy related gene mutation samples. The kit comprises a) uniquely designing and preparing capture probes for ACTC1, ACTN2, BRAF, CALR3, CASQ2, CSRP3, GLA, HRAS, JPH2, KRAS, LAMP2, LDB3, MAP2K1, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYLK2, MYOZ2, PRKAG2, RAF1, SOS1, TCAP, TNNC1, TNNI3, TNNT2, TPM1, TTN, TTR, VCL all exon fragments; b) designing a linker with a unique tag sequence; c) carrying out PCR amplification on the probe sequence by the universal primer; d) unique post-mix target fragment capture operations were designed. The application adopts probe capture sequencing, is the same as other traditional DNA chip hybridization and capillary electrophoresis sequencing technologies, is time-consuming and labor-consuming, is high in price and cannot meet the requirement of gene detection of hypertrophic cardiomyopathy at all.
The next generation sequencing technology is one of the most rapidly developing technologies in the life science field in recent years. In the aspect of genetic disease gene detection, the most remarkable is the next generation of semiconductor sequencing platform, which is released in 2010 by Life Technologies in the United states, is a table type individualized high-throughput gene sequencer specially developed for clinical genetic disease gene detection, takes a high-density semiconductor chip full of micropores as a sequencing basis, and has the characteristics of high speed, economy, good sensitivity, high accuracy and the like.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides a screening method for the mutation of the pathogenic gene related to the hypertrophic cardiomyopathy for non-treatment purposes, which utilizes multiple PCR to amplify target genes, and adopts 314 semiconductor chips to carry out large-scale balanced sequencing on the target genes on the basis of a next generation semiconductor sequencing platform, so that a plurality of case samples can be detected at one time, and the method has the characteristics of simplicity, rapidness, accuracy, economy and the like, and establishes a new method for the clinical early diagnosis and prevention of the hypertrophic cardiomyopathy.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a high throughput screening method for mutations in pathogenic genes associated with hypertrophic cardiomyopathy for non-therapeutic purposes, comprising the steps of:
(1) extracting a genome;
(2) amplifying the target gene by adopting multiplex PCR;
(3) constructing a target gene library;
(4) sequencing the target gene library through a next generation semiconductor sequencing platform, and screening out gene mutation sites related to the occurrence of hypertrophic cardiomyopathy.
In the invention, after the target gene is amplified by utilizing the multiplex PCR, the target gene is subjected to large-scale sequencing by adopting a 314 semiconductor chip on the basis of a next-generation semiconductor sequencing platform, a plurality of case samples can be detected at one time, and the method has the characteristics of simplicity, convenience, rapidness, accuracy, economy and the like.
Preferably, the genome in step (1) is derived from any one or a combination of at least two of human peripheral blood, myocardial tissue, lymphoid organ, spleen, bone marrow or liver.
Preferably, the target gene in step (2) comprises coding regions of all exon fragments of MYH7, MYBPC3, TNNT2, TNNI3, MYH6, TPM1, ACTC1, PRKAG2, MYL2 and MYL3 and non-coding regions of not less than 50bp nearby.
In the present invention, the primer pairs for amplifying coding regions of all exon fragments of target genes MYH7, MYBPC3, TNNT2, TNNI3, MYH6, TPM1, ACTC1, PRKAG2, MYL2 and MYL3 and non-coding regions of not less than 50bp nearby are shown in Table 1 below:
preferably, the reaction system of the multiplex PCR in step (2) is 5 × PrimeSTAR GXL Buffer 10uL, dNTP mix 4uL, 2uL of each of the upstream and downstream primers of 10uM, 2uL (250ng/uL) of template DNA, PrimeSTAR GXLDDNA Polymerase2uL, ddH2O was 30uL and the final volume was 50 uL.
Preferably, the reaction conditions of the multiplex PCR of step (2) are:
(a) pre-denaturation at 98 ℃ for 3 min;
(b) denaturation at 98 ℃ for 10 seconds, annealing at 54 or 57 ℃ for 15 seconds, and extension at 68 ℃ for 1-3 minutes for 35 cycles;
(c) extension at 68 ℃ for 5 minutes.
Preferably, the method further comprises performing multiplex PCR product purification and fragmentation after the multiplex PCR amplification of the target gene in step (2).
Preferably, the multiplex PCR product purification comprises the steps of: the multiplex PCR products of the respective segments of each sample were quantified, mixed in equal proportions, and then purified using magnetic beads.
Preferably, the fragmentation of the multiplex PCR product comprises the steps of: and quantifying the purified product, and breaking the fragment to about 150-250 bp by using a nucleic acid ultrasonication instrument.
Preferably, the semiconductor sequencing platform in step (4) is a 314 semiconductor chip for sequencing.
In the invention, the chips with different fluxes of Life Technologies company are utilized to detect 10 target genes of 7-96 samples at a time, thereby greatly reducing the detection cost of each sample.
Preferably, the method comprises the steps of:
(1) extracting a genome;
(2) amplification of target genes using multiplex PCR:
A) the reaction system of multiplex PCR was 5 × PrimeSTAR GXL Buffer 10uL, dNTP mix 4uL, 2uL for each of the upstream and downstream primers of 10uM, 2uL (250ng/uL) for template DNA, PrimeSTAR GXL DNA Polymerase2uL, ddH2O is 30uL, and the final volume is 50 uL;
B) the reaction conditions for multiplex PCR were: (a) pre-denaturation at 98 ℃ for 3 min; (b) denaturation at 98 ℃ for 10 seconds, annealing at 54 or 57 ℃ for 15 seconds, and extension at 68 ℃ for 1-3 minutes for 35 cycles; (c) extension at 68 ℃ for 5 minutes;
(3) quantifying the multiple PCR products of each section of each sample, mixing the multiple PCR products in equal proportion, purifying by using magnetic beads, quantifying the purified products, and breaking the products to about 150-250 bp by using a nucleic acid ultrasonication instrument;
(4) adding a specific barcode sequence tag joint and a universal sequencing joint to the cut DNA sample;
(5) carrying out electrophoresis recovery on the DNA fragment added with the joint in the step (4) by using high-resolution agarose Gel 2% E-Gel;
(6) constructing a target gene library: carrying out 8-cycle PCR amplification reaction on the DNA fragment recovered by agarose gel electrophoresis in the step (5) by using a universal primer;
(7) sequencing the target gene library through a next-generation semiconductor sequencing platform, and screening out gene mutation sites related to the occurrence of hypertrophic cardiomyopathy: purifying and quantifying the PCR product in the step (6), and sequencing the PCR product by using a next-generation semiconductor sequencing technology platform; and (3) taking the human hg19 genome as a reference genome, and performing quality evaluation and comparison analysis on a sequencing result to find out a possible pathogenic variation site.
In a second aspect, the present invention provides a mutant gene and/or fragment screened by the high-throughput screening method for hypertrophic cardiomyopathy-related pathogenic gene mutation according to the first aspect.
Preferably, the mutant gene and/or fragment is any one or a combination of at least two of MYH7-c.2155c > T, MYH7-c.1987C > T, MYH7-c.2654a > C, MYBPC3-c.706a > G, PRKAG2-c.298g > a and TNNT2-c.887g > a, preferably MYH7-c.2654a > C.
In the present invention, the nucleotide numbering of the isolated mutant gene fragment is carried out according to the following criteria: taking A in the ATG sequence of the initiation codon of the coding region CDS of the gene as +1, for example: MYH7-c.2155C > T, which means that the base C extending to the position of 2155 from the back of the A base in the ATG of the start codon of CDS of the coding region of MYH7 gene is mutated into T, wherein the A base is + 1.
In a third aspect, the present invention provides a nucleic acid fragment comprising a sequence which hybridises to the mutant gene and/or fragment of the second aspect.
Compared with the prior art, the invention has the following beneficial effects:
(1) after the target gene is amplified by utilizing the multiplex PCR, on the basis of a next generation semiconductor sequencing platform, a 314 semiconductor chip is adopted to carry out large-scale sequencing on the target gene, a plurality of case samples can be detected at one time, and the method has the characteristics of simplicity, convenience, rapidness, accuracy, economy and the like, and establishes a new method for the early clinical molecular diagnosis and prevention of hypertrophic cardiomyopathy;
(2) the method can be used for prenatal diagnosis and guidance of prenatal and postnatal care; assisting in definite diagnosis and carrying out clinical intervention; family screening, namely performing family disease occurrence risk assessment and management;
(3) the method is simple, convenient and quick, has low cost, can detect 7-96 samples at one time, lays a foundation for genetic screening of hypertrophic cardiomyopathy, and develops a way for clinical molecular diagnosis.
Drawings
FIG. 1 is an ISP heatmap report for the next generation of semiconductor chip sequencing of the invention;
FIG. 2 is the ISP statistical analysis report for the next generation semiconductor chip sequencing according to the present invention;
FIG. 3 is a histogram report of the sequencing read of the next generation semiconductor chip of the present invention;
FIG. 4 is a report of the comparison analysis of the sequencing result of the next generation semiconductor chip of the present invention with the reference target gene;
FIG. 5 is a report of statistical analysis of sequencing accuracy for next generation semiconductor chips according to the present invention;
FIG. 6 is an IGV analysis of variant sites MYH7-c.2155C > T associated with HCM pathogenesis in an example of the invention and a Sanger sequencing report thereof;
FIG. 7 is an IGV analysis of variant sites PRKAG2-c.298G > A associated with the onset of HCM and a Sanger sequencing report thereof in an example of the present invention;
FIG. 8 is an IGV analysis of variant sites MYH7-c.2654A > C associated with HCM pathogenesis in an example of the invention and a Sanger sequencing report thereof;
FIG. 9 is the IGV analysis and Sanger sequencing report of variant sites MYBPC3-c.706A > G related to HCM pathogenesis in the example of the invention;
FIG. 10 is an IGV analysis of variant sites TNNT2-c.887G > A associated with HCM pathogenesis in an example of the invention and a Sanger sequencing report thereof.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.
Example 1
The method is applied to high-throughput mutation screening of 10 genes (MYH7, MYBPC3, TNNT2, TNNI3, MYH6, TPM1, ACTC1, PRKAG2, MYL2 and MYL3) of 7 hypertrophic cardiomyopathy probands (in addition to collecting the demographic data of HCM patients, the electrocardiograms and the echocardiograms of the 7 hypertrophic cardiomyopathies clinically confirmed in the cardiovascular internal medicine of the first human hospital in Yunnan province), and possible pathogenic mutation sites related to the HCM are searched.
Firstly, genome extraction: for 7 cases of patients with hypertrophic cardiomyopathy confirmed in clinical study, 1ml of peripheral venous blood was collected, after anticoagulation with EDTA, the whole genome was extracted with a commercial Miniprep Kit (Axygen, USA), subjected to agarose gel electrophoresis, and the concentration and OD value were measured, OD260/280Is available in the range of 1.8-2.0.
Secondly, designing a multiplex PCR primer of a target gene: primer design is carried out according to gene accession numbers (MYH 7: NG _007884.1, MYBPC 3: NG _007667.1, TNNT2: NG _007556.1, TNNI3: NG _007866.2, MYH6: NG _023444.1, TPM1: NG _007557.1, ACTC1: NG _007553.1, PRKAG2: NG _007486.1, MYL2: NG _007554.1 and MYL3: NG _007555.2) published by GeneBank database as templates, and the primer coverage range is as follows: the coding region of the exon of the target gene and the base region with two ends not less than 50 bp.
Three, multiple PCR reaction system and optimized condition
1. The preferred PCR amplification reaction system is as follows:
2. preferred PCR reaction conditions are:
fourthly, purification and fragmentation of target gene multiple PCR products
1. The multiplex PCR products of the respective segments of each sample were quantified, mixed in equal proportions, and then purified using AgencourtAmPerure XP Reagent magnetic beads (Invitrogen).
2. The purified product was quantified using a Qubit 2.0 quantifier from Life Technologies, and after quantification, 500ng of the purified PCR fragment was diluted to a final volume of 50ul of nucleic acid-free ddH system 20, carrying out ultrasonic disruption on the fragment by using a CoverisSystems M220 to ensure that the fragment has the size of about 150-250 bp, and freezing the fragment at-20 ℃.
Fifthly, constructing a target gene library: library construction was performed according to the standard protocol for library construction by Life Technologies, Inc., following the following procedure in order to achieve the above-described protocol.
1. End repairing is carried out on the mixed PCR fragment ultrasonically interrupted by the Coveris System M220, 200ul PCR reaction tubes are placed on ice, and 7 fragmented PCR products are prepared according to the following System respectively:
| reagent composition | The dosage of each reaction tube |
| Fragmented DNA fragments | 100ng |
| 5X end repair buffer | 20ul |
| End repair enzyme | 1ul |
| Pure water without nucleic acid | Xul |
| Reaction system | 100ul |
After the preparation, the mixture was mixed uniformly and centrifuged instantaneously using a micropipette, and then incubated for 20 minutes at 25 ℃ on a PCR instrument.
2. And (3) purifying the repaired PCR product: it was purified with Agencour AMPure XP Reagent magnetic beads (Invitrogen) and 25ul of nucleic acid-free ddH was added20, elution was carried out.
3. Linking the adapters and barcode sequence tags, and gap repair.
The following reagent components were prepared in a 200ul PCR reaction tube:
| reagent composition | The dosage of each reaction tube |
| DNA fragment | ～25ul(100ng) |
| 10Xl ligation buffer | 10ul |
| Ion P1adapters | 2ul |
| Ion Xpress Barcode (1-7) sequence tag | 2ul |
| dNTP mixture | 2ul |
| Pure water without nucleic acid | 49ul |
| DNA ligase | 2ul |
| Gap repair polymerases | 8ul |
| Total reaction system | 100ul |
After the preparation is finished, uniformly mixing the components by using a micropipette, and placing the mixture on a PCR instrument for repairing, wherein the reaction conditions are as follows:
| reaction step | Reaction | Reaction time | |
| 1 | 25℃ | 15 minutes | |
| 2 | 72℃ | 5 minutes | |
| 3 | 4℃ | Termination of the reaction |
4. Purification of the ligated and repaired DNA fragments: it was purified with Agencour AMPure XP Reagent magnetic beads (Invitrogen) and finally 20ul of nucleic acid free ddH was added20, elution was carried out.
5. Screening of unamplified library fragment sizes
1) Using a disposable 2% E-Gel (Invitrogen corporation) high resolution electrophoresis Gel, the comb was removed and placed on an iBase Gel electrophoresis apparatus; 2) the first row of the E-Gel was the loading well and the second row was the collection well. Process for screening target fragmentsThe following were used: a) about 20uL of 7 DNA fragments after repair purification were added to each well, 4uL of DNA ladder (500 bp DNA ladder, Shanghai Czeri) was added to the middle well, and 6uL of nucleic acid-free ddH was added to the middle well2O dilute it. 20ul of non-nucleic acid water ddH is respectively added into the collection holes20(DNA ladder corresponding to the collection hole with 10uL of non-nucleic acid water ddH20). b) Collect library fragments of-300 bp: when 300bp DNA ladder runs to the upper reference line of the E-Gel collection well, the electrophoresis is stopped, and 10uL of nucleic acid-free ddH is added to each collection well2And continuing electrophoresis after O. After the target fragment entered the well, the liquid in the corresponding collection well was pipetted into a 200uL PCR reaction tube using a micropipette, followed by 10uL of the nucleic acid-free ddH2O washing the residual DNA in the collection well and sucking it into the corresponding PCR tube.
6. Amplification and purification of unamplified library fragments
And (3) carrying out PCR amplification on the selected PCR library fragment, and preparing an amplification reaction system as follows:
after the PCR reaction was completed, it was purified with Agencour AMPure XP Reagent magnetic beads (Invitrogen Co.), and finally 20ul of nucleic acid-free ddH was added20, elution was carried out.
7. Library quantification: the purified samples were quantified using a Qubit 2.0 quantification apparatus from Life Technologies, and after quantification, the amount of each sample was 26pM, which is calculated as follows:
1.515 Xsample concentration (ng/uL) ÷ library fragment size × 1000 ÷ 26 ═ dilution factor (1uL original library dilution factor)
After diluting the constructed library, 10uL of each library sample is placed in the same 1.5mL EP tube, and after being uniformly mixed, the library is placed at 4 ℃ for standby.
Sixthly, water-in-oil PCR reaction and positive silica gel bead enrichment
To achieve the above purpose, the method is carried out on OT2 and ES instruments of Life Technologies, and comprises the following main steps:
1. water-in-oil PCR reaction: before preparing the amplification reaction solution, a reagent taken out from the temperature of minus 20 ℃ needs to be placed on ice for dissolving, after shaking for 5 seconds, after lightly throwing for 2 seconds, the reagent is reset on the ice, and the PCR amplification reaction solution is prepared, wherein the required reagents and the mixture ratio thereof are as follows:
| serial number | Reagent | Volume of |
| 1 | Ion PGM template OT 2400 mixed reagent | 500uL |
| 2 | Ion PGM template OT 2400 PCR reaction reagent B | 285uL |
| 3 | Ion PGM template OT 2400 PCR polymerase mixture | 50uL |
| 4 | Ion PGM template OT 2400 reagent X | 40uL |
| 5 | 1-7 sample diluted library mixture | 25uL |
| 6 | Ion PGM template OT 2400 silica gel beads | 100uL |
| — | Total reaction system | 1000uL |
After the amplification reaction solution is prepared, the amplification reaction solution is shaken for 5 seconds and uniformly mixed, after the light rate is 2 seconds, the amplification reaction solution is added into a reactor, and then the reactor is placed on an OT2 instrument prepared for water-in-oil PCR reaction to carry out water-in-oil reaction, and the reaction is finished after about 8 hours.
2. Recovering the positive silica gel beads after the amplification reaction
After the water-in-oil PCR reaction was completed, run was pressed down on OT2 instrument, the supernatant and its suspension were slowly aspirated off after centrifugation of the silica gel beads in the collection tubes, leaving about 50uL of each collection tube, incubated at 50 ℃ for 2 minutes, and after enrichment and DNA melting denaturation of silica gel beads containing DNA template with ES instrument, placed at 4 ℃ ready for on-machine sequencing.
Seven, computer sequencing
The PGM instrument was first initialized with buffer and dntps as configured for PGM initialization from life technologies, inc. After successful initialization, on-machine sequencing was performed with reference to the sequencing protocol of Life Technologies, Inc.
Eight, data analysis
1. The analysis was performed by Ion Torrent Server and Ion Report of Life Technologies, Inc.
2. Pathogenicity prediction: the Gene expression was determined by The Human Gene mutation database (HGMD: The Human Gene mutation database,http://www.hgmd.cf.ac.uk/ac/index.php) And the National Center for Biotechnology Information (NCBI: National Center for Biotechnology Information,http:// www.ncbi.nlm.nih.gov/) The SNP database query ofWhether the compound is related to HCM pathogenesis is reported in the literature. If not reported, the mutation sites were applied to the database SIFT according to bioinformatics techniques (http://sift.jcvi.org/)，PolyPhen-2(http://genetics.bwh.harvard.edu/pph2/) And a mutationTaster: (http:// www.mutationtaster.org/) The software predicts whether the mutation site is related to HCM onset. If the mutation sites are related to the onset of HCM, performing Sanger sequencing verification on the mutation sites, and performing case control research on the mutation sites verified to be positive on not less than 100 local normal persons.
Nine results
As a result of the sequencing reaction on 1 314 semiconductor chip, as shown in FIGS. 1 to 5, 88.4M data size was obtained for 7 samples, and 445177 sequences were counted in total, the average length of the sequences was 194bp, the accuracy of reading at 1 Xcoverage of the bases was 99%, and the average depth of sequencing was 31.3X. Of 7 HCM patients, 6 were found to have pathogenic mutation sites: MYH7-c.2155C > T, MYH7-c.1987C > T, MYH7-c.2654A > C, MYBPC3-c.706A > G, PRKAG2-c.298G > A and TNNT2-c.887G > A, wherein MYH7-c.2654A > C is newly found variation sites related to HCM morbidity and is shown in table 1, the rest detection results are shown in figures 6-10, the mutation detection rate is 85.7%, and the accuracy is 100% through Sanger sequencing verification.
TABLE 1 variation sites isolated in association with HCM pathogenesis
In conclusion, the result analysis of the embodiment 1 shows that the invention establishes a simple, convenient, rapid, accurate and economic genetic screening method for the main pathogenic genes of hypertrophic cardiomyopathy.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. A high throughput screening method for the mutation of a pathogenic gene related to hypertrophic cardiomyopathy for non-therapeutic purposes, which is characterized by comprising the following steps:
(1) extracting a genome;
(2) amplifying target genes by using multiplex PCR, wherein the target genes comprise coding regions of all exon fragments of MYH7, MYBPC3, TNNT2, TNNI3, MYH6, TPM1, ACTC1, PRKAG2, MYL2 and MYL3 and non-coding regions which are not less than 50bp nearby;
(3) constructing a target gene library;
(4) sequencing a target gene library through a next-generation semiconductor sequencing platform 314 semiconductor chip, and screening out a gene mutation site related to the incidence of the hypertrophic cardiomyopathy, wherein the gene mutation site is any one or a combination of at least two of MYH7-c.2155C > T, MYH7-c.1987C > T, MYH7-c.2654A > C, MYBPC3-c.706A > G, PRKAG2-c.298G > A and TNNT2-c.887G > A;
the primer of the multiplex PCR is shown as SEQ ID NO 1-172.
2. The high throughput screening method of claim 1, wherein the genome in step (1) is derived from any one or a combination of at least two of human peripheral blood, myocardial tissue, lymphoid organ, spleen, bone marrow or liver.
3. The high throughput screening method of claim 1 or 2, wherein the multiplex PCR in step (2) comprises the reaction system of 5 × PrimeSTAR GXL Buffer 10 μ L, dNTP mix 4 μ L, 10 μ M upstream and downstream primers 2 μ L each, template DNA 2 μ L, the concentration of the template DNA 250ng/μ L, PrimeSTAR GXL DNA Polymerase2 μ L, ddH2O was 30. mu.L and the final volume was 50. mu.L.
4. The method of claim 3, wherein the reaction conditions of the multiplex PCR of step (2) are:
(a) pre-denaturation at 98 ℃ for 3 min;
(b) denaturation at 98 ℃ for 10 seconds, annealing at 54 or 57 ℃ for 15 seconds, and extension at 68 ℃ for 1-3 minutes for 35 cycles;
(c) extension at 68 ℃ for 5 minutes.
5. The high throughput screening method of claim 4, further comprising performing multiplex PCR product purification and fragmentation after the multiplex PCR amplification of the target gene in step (2).
6. The high throughput screening method of claim 5, wherein the multiplex PCR product purification comprises the steps of: the multiplex PCR products of the respective segments of each sample were quantified, mixed in equal proportions, and then purified using magnetic beads.
7. The method of claim 5, wherein the fragmentation of the multiplex PCR product comprises the steps of: and (3) after quantifying the purified product, breaking the fragment into 150-250 bp by using a nucleic acid ultrasonication instrument.
8. The high throughput screening method of claim 1, wherein the method comprises the steps of:
(1) extracting a genome;
(2) amplification of target genes using multiplex PCR:
A) the reaction system of multiplex PCR is 5 × PrimeSTAR GXL Buffer 10. mu.L, dNTP mix 4. mu.L, 10. mu.M upstream and downstream primers 2. mu.L each, template DNA 2. mu.L, the concentration of the template DNA 250 ng/. mu.L, PrimeSTAR GXL DNApolymerase 2. mu.L, ddH2O is 30 μ L, and the final volume is 50 μ L;
B) the reaction conditions for multiplex PCR were: (a) pre-denaturation at 98 ℃ for 3 min; (b) denaturation at 98 ℃ for 10 seconds, annealing at 54 or 57 ℃ for 15 seconds, and extension at 68 ℃ for 1-3 minutes for 35 cycles; (c) extension at 68 ℃ for 5 minutes;
(3) quantifying multiple PCR products of each sample DNA, mixing the multiple PCR products in equal proportion, purifying by using magnetic beads, and finally breaking the multiple PCR products to 150-250 bp by using a nucleic acid ultrasonic crusher;
(4) adding a specific barcode sequence tag joint and a universal sequencing joint to the cut DNA sample;
(5) carrying out electrophoresis recovery on the DNA fragment added with the joint in the step (4) by using high-resolution agarose Gel 2% E-Gel;
(6) constructing a target gene library: carrying out 8-cycle PCR amplification reaction on the DNA fragment recovered by agarose gel electrophoresis in the step (5) by using a universal primer;
(7) sequencing the target gene library through a next-generation semiconductor sequencing platform, and screening out gene mutation sites related to the incidence of hypertrophic cardiomyopathy: purifying and quantifying the PCR product in the step (6), and sequencing the PCR product by using a next-generation semiconductor sequencing technology platform; and (3) taking the human hg19 genome as a reference genome, and performing quality evaluation and comparison analysis on a sequencing result to find out a possible pathogenic variation site.
9. A mutant gene and/or fragment screened by the high-throughput screening method for hypertrophic cardiomyopathy related pathogenic gene mutation of any one of claims 1-8 for non-therapeutic purposes;
the mutant gene and/or fragment is MYH7-c.2654A > C.
10. A nucleic acid fragment comprising a sequence that hybridizes to the mutant gene and/or fragment of claim 9.
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| CN112608994A (en) * | 2020-12-31 | 2021-04-06 | 南昌瑞因康生物科技有限公司 | Detection method, device and storage medium for hypertrophic cardiomyopathy and related genes |
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Effective date of registration: 20231119 Address after: Room 101-1, 9th Floor, Standard Building No. 3, Biotechnology Incubator, Block B-5-28-3, East District, High-tech Zone, Kunming City, Yunnan Province, 650000 Patentee after: Yunnan Kecan Biotechnology Co.,Ltd. Address before: No.727, Jingming South Road, Chenggong University Town, Kunming, Yunnan 650500 Patentee before: Kunming University of Science and Technology |
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