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Primer group, kit and method for detecting gene polymorphism

A gene polymorphism and primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, and microbial assay/inspection, etc., can solve the problem of low throughput of gene polymorphism detection

Active Publication Date: 2021-03-19
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 group, kit and method for detecting gene polymorphism
  • Primer group, kit and method for detecting gene polymorphism
  • Primer group, 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 upstream and downstream primers and corresponding sequencing primers for specific amplification 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 to design primers and analyze dimers and stem-loop mismatches. Primers were designed at both ends containing the site to be tested, and the annealing temperatures of the 10 pairs of primers were basically consistent.

[0083] The primer set provided in this example covers all hotspot gene mutations in APOE gene, CYP4F2 gene, GGCX gene, NPC1L1 gene, NQO1 gene and VKORC1 gene. Since small sequence changes will lead to a significant decrease in primer amplification efficiency and poor specificity, multiple PCR primer sets were designed for different sites / exons, and after pre-experimental screening, the length and position of the product fragmen...

Embodiment 2

[0088] Embodiment 2: DNA is extracted from the sample to be tested

[0089] Step 2.1: Collect oral exfoliated cells with oral 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) to extract DNA from the sample, and use NP80-touch (IMPLEN, Germany) Determine the concentration and purity of DNA, and save the DNA whose test results are within the preset range.

Embodiment 3

[0091] Example 3: Using the primer set synthesized in step 1.2 and the preserved DNA 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 (article number: KFX-101) of TOYOBO Co., Ltd. were used as the basic raw materials. concentration, buffer concentration, and enzyme dosage 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 components 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 amplification / reduction of the reaction system is within the ...

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PUM

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Abstract

The invention provides a primer group, a kit and a method for detecting gene polymorphism. The primer group comprises a primer pair for detecting an APOE gene, a CYP4F2 gene, a GGCX gene, an NPC1L1 gene, an NQO1 gene and a VKORC1 gene. When the primer group is used for performing multiplex PCR detection, mutation of at least one gene can be detected at the same time through one-time PCR reaction,so that the gene detection flux is improved.

Description

technical field [0001] The 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 the formation of blood clots. When vitamin K is deficient, it will cause blood coagulation and abnormal bone metabolism in the body. The APOE gene, CYP4F2 gene, GGCX gene, NPC1L1 gene, NQO1 gene, VKORC1 gene are related to the metabolism of vitamin K. [0003] At present, polymerase chain reaction (PCR) detection tests can be designed for vitamin K metabolism-related genes APOE gene, CYP4F2 gene, GGCX gene, NPC1L1 gene, NQO1 gene and VKORC1 gene to detect gene mutations . [0004] However, since each PCR reaction only targets one exon / mutation site, the detection throughput of gene polymorphism is low. Contents of the invention [0005] The invention provides a primer set, a kit and a method for detecting gene polymorphism, ...

Claims

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Application Information

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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
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