Construction method, primer set and kit of fh sequencing library

A construction method and technology for sequencing libraries, applied in the construction of FH sequencing libraries, primer sets and kits, can solve the problems of low detection throughput, difficulty in applying multi-gene detection, high cost of detection of FH-related gene mutations, and achieve high detection Effects of coverage, low sample consumption, and high uniformity

Active Publication Date: 2022-04-26
深圳安吉康尔医学检验实验室
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Problems solved by technology

[0012] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art, and provide a method for constructing an FH sequencing library, a primer set and a kit, aiming at solving the problems of high detection cost, low detection throughput, and difficulty in applying existing FH-related gene mutations. Technical issues of multigene testing for complex diseases like FH

Method used

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  • Construction method, primer set and kit of fh sequencing library
  • Construction method, primer set and kit of fh sequencing library
  • Construction method, primer set and kit of fh sequencing library

Examples

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Embodiment 1

[0058] It is known that the sample S1 of a clinically diagnosed patient with familial hypercholesterolemia is LDLR gene mutation verified by whole-exome high-throughput sequencing and Sanger sequencing gold standard, and is detected by the embodiment of the present invention.

[0059] Main instruments: Eppendorf centrifuge 5424, Shanghai Yi constant temperature metal bath TUS-200P, ABI thermal cycler Veriti Dx, ABI fluorescence quantitative instrument 7500, Illumina high-throughput sequencer Miseq, etc.

[0060] 1. High-throughput sequencing library construction, such as figure 1 shown, including the following steps:

[0061] 1) Nucleic acid DNA extraction

[0062] For the extraction of nucleic acid DNA from human whole blood samples, peripheral blood nucleic acid extraction was carried out according to Beijing Tiangen's blood genome DNA extraction kit, and DNA samples were obtained for future use.

[0063] 2)1 st PCR amplification (multiple PCR amplification) and purifica...

Embodiment 2

[0080] It is known that the sample S2 of the clinically diagnosed patient with familial hypercholesterolemia is verified as LDLR and APOE gene mutations by whole-exome high-throughput sequencing and Sanger sequencing gold standard, and is detected by the present invention. The detection method is the same as in Example 1.

[0081] The test results found that a possible pathogenic heterozygous mutation 1879G>A (p.Ala627Thr) was found in the exon 13 region of the LDLR gene, and a heterozygous mutation c that increased the risk of disease was found in the exon 4 region of the APOE gene .388T>C(p.Cys130Arg), and the hotspot mutation sites of other genes were all negative, and the detection results were consistent with the results of whole exome sequencing and Sanger verification.

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Abstract

The invention belongs to the field of gene sequencing, and in particular relates to a method for constructing an FH sequencing library, a primer set and a kit. The method for constructing the FH sequencing library includes the following steps: obtaining a DNA sample; performing multiple PCR amplification on the DNA sample by using the first primer set and the second primer set, respectively, to obtain a first amplification product and a second amplification product; Mixing the first amplification product and the second amplification product to obtain a mixed product; amplifying the mixed product with sequencing adapter primers to obtain a sequencing library; wherein, the sequence of the first primer set is as SEQ ID NO.1- 130; the sequence of the second primer set is shown in SEQ ID NO.131-260. The construction method of the FH sequencing library has the advantages of high detection coverage and high uniformity, less sample consumption, low sample requirements and a large saving in detection costs, and can provide a technical basis for clinical discovery of new gene mutations.

Description

technical field [0001] The invention belongs to the field of gene sequencing, and in particular relates to a method for constructing an FH sequencing library, a primer set and a kit. Background technique [0002] Familial hypercholesterolemia (familial hypercholesterolemia, FH) is a genetic disease of metabolic abnormalities. The LDL-C level of patients can reach 190mg / dL~400mg / dL, or even higher. High levels of LDL-C are the core determinants of the occurrence and development of atherosclerotic cardiovascular diseases (ASCVD), such as coronary heart disease, myocardial infarction, and ischemic stroke. Among the several major factors that cause LDL-C to rise, unhealthy eating habits and lifestyle are two important factors, but genetic factors also play a key role. Studies have found that the liver of FH patients cannot normally metabolize excessive low-density lipoprotein (lowdensity lipoprotein, LDL), which is precisely caused by pathogenic mutations in genes related to L...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12Q1/6806C12Q1/6869C12N15/11
CPCC12Q1/6806C12Q1/6869C40B50/06
Inventor 李妍珂李全陈玥茏刘永初刘阳李阳吕佩涛
Owner 深圳安吉康尔医学检验实验室
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