Kit for detecting polymorphism of APOE gene and SLCO1B1 gene

Abstract

The invention provides a kit for detecting polymorphism of an APOE gene and an SLCO1B1 gene. The kit comprises primers used for detecting polymorphism sites T388C and C526T of the APOE gene and expressed as SEQ ID NO. 1-6, primers used for detecting polymorphism sites A388G and T521C of the SLCO1B1 gene and expressed as SEQ ID NO. 7-12, and internal quality control housekeeping gene primers expressed as SEQ ID NO. 13-20. The kit is used for carrying out fluorescence PCR by using ARMS-PCR and fluorescence fusion curve techniques, is capable of achieving accurate detection of one polymorphism oftwo genes in the same reaction tube, is high in sensitivity and high in specificity, and can meet detection of oral epithelial cells, dried-blood spots and whole-blood samples; the whole detection process is short in time; the medication basis can be provided for the doctor in the first time; the medication risks of the patients can be reduced.

Description

technical field

[0001] The invention relates to the field of molecular biology, in particular to a detection kit for APOE and SLCO1B1 gene polymorphism. Background technique

[0002] The APOE gene is a gene encoding human apolipoprotein E (apolipoprotein E, APOE). This gene is located on chromosome 19p13 and has 4 exons and 3 introns. The APOE protein encoded by it belongs to the apolipoprotein family. It is an important part of plasma lipoprotein, mainly expressed in liver and brain tissue, participates in the body's lipid metabolism through various channels, and is an important internal factor affecting blood lipid levels.

[0003] Studies have shown that the APOE gene has nucleotide polymorphisms, and the two most common single nucleotide polymorphisms are APOE T388C (rs429358) and APOE C526T (rs7412), forming three haploids, respectively For E2 (388T-526T), E3 (388T-526C), E4 (388C-526C) three protein phenotypes, including three homozygous (E2 / E2, E3 / E3, E4 / E4) and thre...

Examples

Embodiment 1

[0085] Example 1 Optimization experiment of primers and quality control housekeeping gene primers for detection of APOE and SLCO1B1 gene polymorphisms

[0086]The present invention designs multiple pairs of ARMS primers, and compares the detection efficiencies of the primers, that is, the comparison of the Ct values ​​of the PCR amplification curves. The smaller the Ct value, the higher the efficiency of the primers and the higher the detection sensitivity. The primers suffixed with D in Table 5 are another primer designed for the same detection gene locus. The housekeeping gene primer suffixed with D is another designed primer. The downstream primers at the same site are the same primer.

[0087] table 5

[0088]

[0089]

[0090] When the reaction system is formed, the present invention has 4 reaction systems, and each system has 6 primers. If there is no optimization and improvement between the primers, there will inevitably be pairing or other mismatches between th...

Embodiment 2

[0127] Embodiment 2 Assembly of APOE and SLCO1B1 gene polymorphism detection kit of the present invention

[0128] 1. Primer synthesis: specific primers determined in Example 1. The primers were prepared as 100uM stock solution for storage.

[0129] 2. Preparation of internal reference system: 4 pairs of internal reference primers designed for the human genome determined in Example 1, the primer pair sequences are ACTB-1F and ACTB-1R, ACTB-2F and ACTB-2R, ACTB-3F and ACTB-3R, ACTB -4F and ACTB-4R; the internal reference primers were respectively prepared into 100 μM mother solution for storage.

[0130] 3. Prepare a positive control substance and a blank control substance. The positive control substance contains 4 kinds of plasmid DNA, and these plasmid DNAs contain APOE 388T, APOE 388C, APOE 526C, APOE 526T, SLCO1B1 388A, SLCO1B1 388G, SLCO1B1 521T, SLCO1B1 521C , the internal reference plasmid ACTB of the housekeeping gene, the selection and design of this plasmid are well...

Embodiment 3

[0138] The APOE and SLCO1B1 gene polymorphism detection kit prepared in Example 2 was used to detect the samples to be tested. 1. Prepare the reaction mixture (Mix) for each site, and prepare according to the table below;

[0139] Table 9

[0140] Element

per reaction volume

N share system

2X Master mix

12.5ul

N×12.5ul

add water

10.5ul

N×10.5ul

[0141] 2. Fluorescent quantitative PCR detection of samples

[0142] Take 2 μl of the extracted qualified DNA samples and add them to the 23 μl Mix1, Mix2, Mix3, and Mix4 detection reaction systems of the kit in Example 2, so that the total volume of the four reaction systems is 25 μl, and put them into the fluorescence quantitative PCR instrument, set the PCR reaction program as follows to carry out the amplification reaction:

[0143] 50°C for 2min; 95°C for 2min; 95°C for 10s, 56°C for 45s, 45 cycles; collect the fluorescence signal of SYBR after each cycle.

[0144] Melting cur...

technical field

[0001] The invention relates to the field of molecular biology, in particular to a detection kit for APOE and SLCO1B1 gene polymorphism. Background technique

[0002] The APOE gene is a gene encoding human apolipoprotein E (apolipoprotein E, APOE). This gene is located on chromosome 19p13 and has 4 exons and 3 introns. The APOE protein encoded by it belongs to the apolipoprotein family. It is an important part of plasma lipoprotein, mainly expressed in liver and brain tissue, participates in the body's lipid metabolism through various channels, and is an important internal factor affecting blood lipid levels.

[0003] Studies have shown that the APOE gene has nucleotide polymorphisms, and the two most common single nucleotide polymorphisms are APOE T388C (rs429358) and APOE C526T (rs7412), forming three haploids, respectively For E2 (388T-526T), E3 (388T-526C), E4 (388C-526C) three protein phenotypes, including three homozygous (E2 / E2, E3 / E3, E4 / E4) and thre...

Examples

Embodiment 1

[0085] Example 1 Optimization experiment of primers and quality control housekeeping gene primers for detection of APOE and SLCO1B1 gene polymorphisms

[0086]The present invention designs multiple pairs of ARMS primers, and compares the detection efficiencies of the primers, that is, the comparison of the Ct values ​​of the PCR amplification curves. The smaller the Ct value, the higher the efficiency of the primers and the higher the detection sensitivity. The primers suffixed with D in Table 5 are another primer designed for the same detection gene locus. The housekeeping gene primer suffixed with D is another designed primer. The downstream primers at the same site are the same primer.

[0087] table 5

[0088]

[0089]

[0090] When the reaction system is formed, the present invention has 4 reaction systems, and each system has 6 primers. If there is no optimization and improvement between the primers, there will inevitably be pairing or other mismatches between th...

Embodiment 2

[0127] Embodiment 2 Assembly of APOE and SLCO1B1 gene polymorphism detection kit of the present invention

[0128] 1. Primer synthesis: specific primers determined in Example 1. The primers were prepared as 100uM stock solution for storage.

[0129] 2. Preparation of internal reference system: 4 pairs of internal reference primers designed for the human genome determined in Example 1, the primer pair sequences are ACTB-1F and ACTB-1R, ACTB-2F and ACTB-2R, ACTB-3F and ACTB-3R, ACTB -4F and ACTB-4R; the internal reference primers were respectively prepared into 100 μM mother solution for storage.

[0130] 3. Prepare a positive control substance and a blank control substance. The positive control substance contains 4 kinds of plasmid DNA, and these plasmid DNAs contain APOE 388T, APOE 388C, APOE 526C, APOE 526T, SLCO1B1 388A, SLCO1B1 388G, SLCO1B1 521T, SLCO1B1 521C , the internal reference plasmid ACTB of the housekeeping gene, the selection and design of this plasmid are well...

Embodiment 3

[0138] The APOE and SLCO1B1 gene polymorphism detection kit prepared in Example 2 was used to detect the samples to be tested. 1. Prepare the reaction mixture (Mix) for each site, and prepare according to the table below;

[0139] Table 9

[0140] Element

per reaction volume

N share system

2X Master mix

12.5ul

N×12.5ul

add water

10.5ul

N×10.5ul

[0141] 2. Fluorescent quantitative PCR detection of samples

[0142] Take 2 μl of the extracted qualified DNA samples and add them to the 23 μl Mix1, Mix2, Mix3, and Mix4 detection reaction systems of the kit in Example 2, so that the total volume of the four reaction systems is 25 μl, and put them into the fluorescence quantitative PCR instrument, set the PCR reaction program as follows to carry out the amplification reaction:

[0143] 50°C for 2min; 95°C for 2min; 95°C for 10s, 56°C for 45s, 45 cycles; collect the fluorescence signal of SYBR after each cycle.

[0144] Melting cur...

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