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Primer set, kit and method for detecting gene polymorphism

A gene polymorphism, primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc. Intuitive and accurate interpretation, the effect of reducing inspection costs

Active Publication Date: 2022-07-19
济南和合医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, since each PCR reaction only targets one exon / mutation site, the detection throughput of genetic polymorphisms is low

Method used

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  • Primer set, kit and method for detecting gene polymorphism
  • Primer set, kit and method for detecting gene polymorphism
  • Primer set, kit and method for detecting gene polymorphism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Design and synthesis of primer sets

[0081] Step 1.1: Design specific amplification upstream and downstream primers and corresponding sequencing primers based on APOE gene, CYP4F2 gene, GGCX gene, NPC1L1 gene, NQO1 gene and VKORC1 gene.

[0082] For the designed primers, Primer Quest and Primer Premier 5.0 were used for primer design and dimer and stem-loop mismatch analysis, and primers were designed at both ends containing the sites to be tested. The annealing temperatures of the 10 pairs of primers were basically the same.

[0083] The primer set provided in this example covers all hot spot gene mutations of APOE gene, CYP4F2 gene, GGCX gene, NPC1L1 gene, NQO1 gene and VKORC1 gene. Since small sequence changes will significantly reduce the amplification efficiency and specificity of primers, multiple PCR primer sets are designed for different loci / exons, and after pre-experiment screening, the length and position of the product fragments are integrated. ...

Embodiment 2

[0088] Example 2: DNA extraction from samples to be tested

[0089] Step 2.1: Collect buccal exfoliated cells with buccal swabs or collect fresh peripheral blood samples with blood collection tubes.

[0090] Step 2.2: Use Tiangen Oral Swab Genomic DNA Extraction Kit (DP322), or, use Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304), extract DNA from the sample, and use NP80-touch (IMPLEN, Germany) Determine the concentration and purity of DNA, and save DNA with test results within a preset range.

Embodiment 3

[0091] Example 3: Using the primer set synthesized in step 1.2 and the DNA stored in step 2.2 to prepare a PCR reaction system

[0092] Step 3.1: Use the genomic DNA saved in step 2.2 as the amplification template, and use the primer set synthesized in step 1.2 to prepare a multiplex PCR reaction system.

[0093] In this example, the DNA polymerase and buffer in the KOD FX enzyme system (Item No.: KFX-101) of TOYOBO Co., Ltd. are used as the basic raw materials. The concentration, buffer concentration, and enzyme dosage are used to prepare a multiplex PCR amplification system. The specific composition of this reaction system is shown in Table 3 below.

[0094] table 3

[0095] Reagent ingredients volume 2×PCR buffer for FX 25μl 2mM dNTPs 12.5μl Primer Mix 8.25μl KOD FX (1U / μl) 1.25μl DNA 2μl Ultra-pure water 1μl

[0096] It should be noted that the proportional enlargement / reduction of the reaction system is within th...

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PUM

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Abstract

The present invention provides a primer set, a kit and a method for detecting gene polymorphism. The primer set includes: a primer pair for detecting APOE gene, CYP4F2 gene, GGCX gene, NPC1L1 gene, NQO1 gene and VKORC1 gene. When using the primer set for multiplex PCR detection, the mutation of at least one gene can be simultaneously detected by one PCR reaction, so that the gene detection throughput is improved.

Description

technical field [0001] The present invention relates to the technical field of gene detection, in particular to a primer set, a kit and a method for detecting gene polymorphism. Background technique [0002] Vitamin K is an essential fat-soluble vitamin for blood clot formation. When vitamin K is deficient, it will cause coagulation and abnormal bone metabolism in the body. The APOE gene, CYP4F2 gene, GGCX gene, NPC1L1 gene, NQO1 gene, VKORC1 gene were related to vitamin K metabolism. [0003] At present, polymerase chain reaction (PCR) detection tests can be designed for the vitamin K metabolism-related genes APOE gene, CYP4F2 gene, GGCX gene, NPC1L1 gene, NQO1 gene and VKORC1 gene, respectively, to detect gene mutations. . [0004] However, since each PCR reaction only targets one exon / mutation site, the detection throughput of gene polymorphism is low. SUMMARY OF THE INVENTION [0005] The present invention provides a primer set, a kit and a method for detecting gene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2537/143C12Q2565/125
Inventor 冯薇赵方圆智慧芳倪君君
Owner 济南和合医学检验有限公司