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A method for constructing a gene detection library for familial hypercholesterolemia and its kit

A technology for hypercholesterolemia and gene detection, which is applied in chemical libraries, biochemical equipment and methods, and microbial measurement/testing, and can solve problems such as increasing manual operations

Active Publication Date: 2020-09-04
ANNGEEN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fragmentation of DNA by physical fragmentation requires investment in the fragmentation instrument and instrument maintenance, and the enrichment of DNA fragments must be purified by magnetic beads before the subsequent steps of library construction can be carried out, which further increases manual operations

Method used

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  • A method for constructing a gene detection library for familial hypercholesterolemia and its kit
  • A method for constructing a gene detection library for familial hypercholesterolemia and its kit
  • A method for constructing a gene detection library for familial hypercholesterolemia and its kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] 16 samples were used for library construction, multiple reaction PCR was performed using the primer pool containing the full coding region of LDLR, APOB, and PCSK9 genes and the variable splicing region (20 bp extension from exon to intron) as the target region, and combined The high-throughput sequencing platform S5 Plus performs DNA sequencing, and then detects point mutations (SNPs) and small insertion deletions (InDels). The specific operation process is as follows:

[0083] (1) Nucleic acid extraction and quality inspection: After blood cells undergo nucleic acid extraction and quality inspection, they are required to meet certain quality control standards: DNA concentration: 5ng / μL; DNA purity: OD260 / 280 1.8-2.0, OD260 / 230>2; DNA Total starting amount: 15ng;

[0084] (2) Multiplex PCR amplification: In order to ensure that the target region (the entire coding region of the gene to be tested, and the variable splicing region) is fully covered, and to avoid the for...

Embodiment 2

[0097] In this example, 16 samples were used for library construction, and multiple reaction PCR was performed using the primer pool containing the entire coding region of LDLR, APOB, and PCSK9 genes and the variable splicing region (20 bp extension from exon to intron) as the target region , and combined with the high-throughput sequencing platform Ion Torrent PGM for DNA sequencing, and then detected point mutations (SNPs) and small insertion-deletions (InDels). The specific operation process is as follows:

[0098] (1) Nucleic acid extraction and quality inspection: After blood cells undergo nucleic acid extraction and quality inspection, they are required to meet certain quality control standards: DNA concentration: 5ng / μL; DNA purity: OD260 / 280 1.8-2.0, OD260 / 230>2; DNA Total starting amount: 15ng;

[0099] (2) Multiplex PCR amplification: In order to ensure that the target region (the entire coding region of the gene to be tested, and the variable splicing region) is fu...

Embodiment 3

[0115] In this example, 16 samples were used for library construction, and multiple reaction PCR was performed using the primer pool containing the entire coding region of LDLR, APOB, and PCSK9 genes and the variable splicing region (20 bp extension from exon to intron) as the target region , and combined with the high-throughput sequencing platform Ion Torrent PGM for DNA sequencing, and then detected point mutations (SNPs) and small insertion-deletions (InDels). The specific operation process is as follows:

[0116] (1) Nucleic acid extraction and quality inspection: After nucleic acid extraction and quality inspection of oral mucosal exfoliated cells, they are required to meet certain quality control standards: DNA concentration: 5ng / μL; DNA purity: OD260 / 280 1.8-2.0, OD260 / 230> 2; DNA total initial amount: 15ng;

[0117] (2) Multiplex PCR amplification: In order to ensure that the target region (the entire coding region of the gene to be tested, and the variable splicing ...

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Abstract

The invention discloses a construction method of a gene detection library of familial hypercholesterolemia and a gene detection kit and relates to gene mutation of LDLR (Low-Density Lipoprotein Receptor), APOB (Apolipoprotein B) and PCSK9 (Proprotein Convertase Subtilisin / Kexin type 9). In order to ensure that all target regions (including an entire coding region and an alternative splicing regionof a gene to be detected) are covered and prevent a primer dimer or a short segment from being formed between primers of adjacent amplicons, a PCR (Polymerase Chain Reaction) amplification primer isdivided into two independent primer pools; then primer sequence digestion, sequencing connector connection, library purification and quality detection are carried out; finally, the detection library is constructed. The method has the advantages that library establishment steps are simple and rapid, the cost is effectively reduced, the working amount is reduced, variation types are comprehensive, the detection accuracy reaches 100 percent and the flux is high.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and kit for constructing a gene detection library for familial hypercholesterolemia. Background technique [0002] Familial Hypercholesterolemia (FH) is a rare autosomal dominant genetic disease with an incidence of 1 / 500 in the population. It is divided into two subtypes according to the genotype, and the homozygous family Hypercholesterolemia (HoFH) and heterozygous familial hypercholesterolemia (HeFH). The low-density lipoprotein (LDL) cholesterol value of the patient itself is abnormally high. If the patient is a homozygous patient, the low-density lipoprotein (LDL) cholesterol value is 4 to 6 times that of a normal person, and the LDL-C is usually 500-1200mg / dL , even more than 700mg / dL. Hypercholesterolemia is a predisposing factor for heart disease. People with FH and untreated people are about 20 times more likely to suffer from coronary heart disease th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12N15/10C40B50/06
CPCC12N15/1093C12Q1/6806C40B50/06C12Q2531/113C12Q2537/143C12Q2525/191
Inventor 周洋扶媛媛曹彦东马懿杨颖
Owner ANNGEEN BIOTECHNOLOGY CO LTD
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