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Primers, kit and pcr method for detecting polymorphism of apoe gene

A gene polymorphism and kit technology, applied in the fields of biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of inability to detect clinical specimens on a large scale at the same time, high price of testing instruments, and low clinical popularity. and other problems to achieve the effect of avoiding site mismatches, fast detection, and good sensitivity

Active Publication Date: 2017-09-19
沈阳优吉诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0019] In view of this, the present invention provides a kind of primer that detects ApoE gene polymorphism, test kit and its PCR method, to at least solve the complex interpretation of the result that exists in the past kit, the detection instrument price is high, the operation difficulty is big, there is certain false negative One or more problems such as false positives, high testing costs, low clinical popularity, and inability to test clinical specimens on a large scale at the same time

Method used

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  • Primers, kit and pcr method for detecting polymorphism of apoe gene
  • Primers, kit and pcr method for detecting polymorphism of apoe gene
  • Primers, kit and pcr method for detecting polymorphism of apoe gene

Examples

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Effect test

Embodiment 1

[0133] Example 1: Preparation of wild type and mutant positive plasmids for ApoE gene polymorphism detection

[0134] The human ApoE gene is located on the long arm of chromosome 19, region 13, band 2 (19q132). The full length of the gene is 3.7kb, containing 4 exons and 3 introns, and its cDNA length is 1.163kb. The precursor of ApoE protein consists of 317 amino acids and contains 18 amino acid signal peptides, and the mature ApoE protein consists of 299 amino acids. The ApoE gene has two SNPs, rs429358 and rs7412 located at codons 112 (c.388 T>C) and 158 (c.526 C>T), respectively.

[0135] First, we called out the gene sequence before and after the rs429358 site of the ApoE gene from the gene bank, and marked the polymorphic site with a double underline, at the appropriate position upstream and downstream of the rs429358 site of the ApoE gene (marked in bold and underlined), Design a pair of cloning primers, the amplified fragment is 218bp, including the polymorphic site, ...

Embodiment 2

[0160] Example 2: Design and specificity screening of allele-specific primers (ASP)

[0161] For the rs429358 site of the ApoE gene, wild-type and a series of mutant-specific primers were designed as follows:

[0162] ApoE-rs429358-WT-F: cgcggacatggaggacgagt (SEQ No. 15)

[0163] ApoE-rs429358-mut-F: cgcggacatggaggacgagc (SEQ No. 16)

[0164] ApoE-rs429358-mut-F1:ggacatggaggacgtcc (SEQ No. 17)

[0165] ApoE-rs429358-mut-F2: cggacatggaggacgagc (SEQ No. 18)

[0166] ApoE-rs429358-mut-F3: gcggacatggaggacgtcc (SEQ No. 19)

[0167] ApoE-rs429358-mut-F4: cggacatggaggacgtcc (SEQ No. 20)

[0168] ApoE-rs429358-mut-F5: gcggacatggaggacgagc (SEQ No. 21)

[0169] At the same time, a Taqman-specific probe (SEQ No.22) was designed and synthesized: FAM-ccgcggcgaggtgcaggccatgc-BHQ1. Relevant primers and probes were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

[0170] Then use the above 7 primers to pair with ApoE-rs429358-R (SEQ No.23): 5'-cgcagctcctcggtgctctg-3' primers,...

Embodiment 3

[0172] Embodiment 3: ASP sensitivity screening

[0173] We then paired the mutant primers with the ApoE-rs429358-R primers, and used the mutant recombinant plasmids according to 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10, 0 for serial dilution, plus Taqman-specific probes, sensitivity verification was performed on a fluorescent quantitative PCR instrument. Mutant-specific primer No. 7 can detect 100 copies of the mutant, so this primer is the best primer for detecting ApoE polymorphic sites screened according to our method, as shown in Table 3 for details.

[0174] According to the same method, we designed and screened ApoE rs7412 site-specific mutation and wild-type primers, common downstream primers and probes, as shown in Table 3.

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Abstract

The invention discloses primers for detecting ApoE gene polymorphism, a kit and a PCR (polymerase chain reaction) method for the primers or the kit. The primers include two sets of primers for detecting an rs429358 site and an rs7412 site respectively; the first set of primers include a mutant-type specific upstream primer of the rs429358 site, a wild-type specific upstream primer of the rs429358 site and a first downstream primer shared by the mutant-type specific upstream primer and the wild-type specific upstream primer of the rs429358 site; the second set of primers include a mutant-type specific upstream primer of the rs7412 site, a wild-type specific upstream primer of the rs7412 site and a second downstream primer shared by the mutant-type specific upstream primer and the wild-type specific upstream primer of the rs7412 site. The kit has the advantages of simplicity, quickness, accuracy and low price for detection and the like, and provides a powerful tool for scientific research and clinical analysis of ApoE gene typing and gene mutation.

Description

technical field [0001] The invention relates to the field of molecular biological gene detection, and in particular provides primers, a kit and a PCR method for detecting ApoE gene polymorphisms, which are used for rapid detection of two polymorphic sites of ApoE gene rs429358 and rs7412. Background technique [0002] The ApoE gene is closely related to a variety of cardiovascular and cerebrovascular diseases. The encoded apolipoprotein E (Apolipoprotein E, ApoE) is involved in the entire occurrence and development of cardiovascular and cerebrovascular diseases such as hyperlipidemia, atherosclerosis, and coronary heart disease. Morality is the main cause of individual differences in the early stages and development of these diseases. [0003] The exchange of two amino acid residues, arginine (Arg) and cysteine ​​(Cys) at positions 112 and 158 of the amino acid sequence of ApoE determines the type of isomer. There are three isoforms of the ApoE gene, ApoE2, ApoE3 and ApoE4,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2531/113C12Q2535/125C12Q2561/101
Inventor 高劲松张英杰李星颐刘会影魏潇魏奇
Owner 沈阳优吉诺生物科技有限公司
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