Construction method of gene detection library of hereditary hypertrophic cardiomyopathy and kit
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A technology for hypertrophic cardiomyopathy and gene detection, which is applied in chemical libraries, biochemical equipment and methods, and microbial determination/inspection.
Inactive Publication Date: 2019-03-19
ANNGEEN BIOTECHNOLOGY CO LTD
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If the multiplex PCR method is used to amplify the target region during the sample library preparation process, there will be am...
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Embodiment 1
[0103] Four peripheral blood samples were used for library construction, using the full coding region and variable splicing region (exon to intron extension) of ACTC1, ACTN2, MYBPC3, MYH7, MYL2, MYL3, TNNI3, TNNT2, TPM1 genes 20bp) as the primer pool of the target region for multiplex reaction PCR, combined with the high-throughput sequencing platform Miseq for DNA sequencing, and then detected point mutations (SNP) and small fragment insertion-deletion (InDel). The specific operation process is as follows:
[0104] (1) Nucleic acid extraction and quality inspection: After nucleic acid extraction and quality inspection of peripheral blood samples, it is required to meet certain quality control standards: DNA integrity is good, no protein RNA pollution, DNA concentration ≥ 20ng / μL; DNA purity: OD260 / 280 1.8-2.0, OD260 / 230>2.0; total DNA input amount: 0.1μg-1μg.
[0105](2) Library preparation: Using the above DNA, make up to 52.5 μl with TRIS-EDTA after sampling, perform ultr...
Embodiment 2
[0113] 16 buccal swab samples were used for library construction, using the full coding region and variable splicing region (exon to intron extension) of ACTC1, ACTN2, MYBPC3, MYH7, MYL2, MYL3, TNNI3, TNNT2, TPM1 genes 20bp) as the primer pool of the target region for multiplex reaction PCR, combined with the high-throughput sequencing platform Nextseq550AR for DNA sequencing, and then detecting point mutations (SNPs) and small fragment insertions and deletions (InDels). The specific operation process is as follows:
[0114] (1) Nucleic acid extraction and quality inspection: After nucleic acid extraction and quality inspection of peripheral blood samples, it is required to meet certain quality control standards: DNA integrity is good, no protein RNA pollution, DNA concentration ≥ 20ng / μL; DNA purity: OD260 / 280 1.8-2.0, OD260 / 230>2.0; total DNA input amount: 0.1μg-1μg.
[0115] (2) Library preparation: Using the above DNA, make up to 52.5 μl with TRIS-EDTA after sampling, pe...
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Abstract
The invention discloses a construction method of a gene detection library of hereditary hypertrophic cardiomyopathy and a kit and relates to mutation of ACTC1, ACTN2, MYBPC3, MYH7, MYL2, MYL3, TNNI3,TNNT2 and TPM1 genes. Target regions comprise an exon region of coding amino acid of the 9 genes and 20 basic group regions at the upstream and downstream of an exon; in order to ensure that all the target regions are covered, a target region library is obtained through a hybridization probe capturing method after the library is prepared; then an LMPCR (Ligation-Mediated Polymerase Chain Reaction)method is adopted to amplify the library; and after the library is purified, a sample library for sequencing is obtained. The library construction method has simple steps and a rapid speed and the cost of constructing the library is effectively reduced; related genes of the hypertrophic cardiomyopathy are covered; an illumina high-throughput sequencing machine is combined and related gene mutation can be rapidly and accurately obtained; and the construction method has great significance on the hereditary hypertrophic cardiomyopathy.
Description
technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and a kit for constructing a gene detection library of hereditary hypertrophic cardiomyopathy. Background technique [0002] Hereditary hypertrophic cardiomyopathy (HCM) is a relatively common monogenic cardiovascular disease and one of the main causes of sudden death in adolescents. damage. Hereditary hypertrophic cardiomyopathy occurs in approximately 1 in 500, but due to the presence of asymptomatic carriers of HCM pathogenic variants, the true prevalence may be as high as 1 in 200. [0003] The main clinical symptoms of HCM include shortness of breath, chest tightness, palpitation, palpitations, amaurosis and syncope. Most patients develop cardiac hypertrophy in adolescence and middle age, but it also occurs in infancy and old age. HCM is a progressive disease with different clinical stages, ranging from asymptomatic to extremely severe left ventricular rem...
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