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Primers and kits for detecting human apoc3 gene mutation

A technology of APOC3-PF5 and APOC3-PR5, which is applied in the field of molecular biology, can solve the problems of sample contamination, increased detection cost, and increased detection steps, etc.

Active Publication Date: 2017-12-05
浙江绍兴鼎晶生物医药科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are some substances that inhibit the PCR reaction in the whole blood. Generally, human genomic DNA needs to be extracted before the PCR reaction can be performed. This requires the purchase of an additional DNA extraction kit, which increases the cost of detection and increases the detection steps, which is likely to cause sample contamination.

Method used

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  • Primers and kits for detecting human apoc3 gene mutation
  • Primers and kits for detecting human apoc3 gene mutation
  • Primers and kits for detecting human apoc3 gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Whole blood direct PCR, PCR with blood extraction genomic DNA as template, and PCR with pUC19-APOC3 plasmid as template result comparison

[0033] 1. Design primers

[0034] APOC3 gene-specific primer sequences are as follows:

[0035]Forward primer: APOC3-PF 5'-GACCCAGCTAAGGTTCTAC-3', as shown in SEQ ID No.3, reverse primer: APOC3-PR 5'-AGGCTGAAGAGGCACAG-3', as shown in SEQ ID No.4. The corresponding PCR product size is 559bp. The primers were synthesized by Suzhou Jinweizhi Company.

[0036] 2. Extract blood genomic DNA

[0037] According to the instructions of Qiagen Blood DNA Extraction Kit (Product No. 158422), 200ul of freshly collected blood can obtain 1-5ug of human whole genome DNA, diluted to 100ng / ul for use, and stored at -20°C for a long time.

[0038] 3. Use the extracted plasmid containing wild-type APOC3 gene and mutant APOC3 gene as a template as a control for whole blood PCR: whole gene synthesis of wild-type APOC3 gene, sequence 1, and ...

Embodiment 2

[0056] Example 2: PCR of blood samples from different anticoagulation sources and different blood concentrations

[0057] 1. Collect whole blood

[0058]Fresh blood from the same person was collected and collected in EDTA anticoagulant tubes, sodium heparin anticoagulant tubes and sodium citrate anticoagulant tubes, each numbered as A, B, and C.

[0059] 2. Preparation of PCR reaction solution

[0060] Prepare the PCR reaction solution as follows:

[0061] Concentration 1: 2 μul whole blood was added to every 20 μul reaction solution, as shown in Table 3.

[0062] table 3

[0063] Element

Reaction Tube A1

Reaction tube B1

Reaction tube C1

template whole blood

2μl

2μl

2μl

2×Taq Master Mix

10μl

10μl

10μl

Primer APOC3-PF / APOC3-PR

1-5pMol

1-5pMol

1-5pMol

ddH2O

up to 20ul

up to 20ul

up to 20ul

[0064] Concentration 2: Add 4 μul whole blood for every 20 μul reaction solution, a...

Embodiment 3

[0076] Embodiment 3: the impact of different PCR reaction procedures on experimental results

[0077] Because, although the experimenters set the same PCR program on the PCR machine, the heating and cooling speeds of different PCR machines will be different, which will lead to differences in the reaction time of each temperature, thereby affecting the PCR results. The key step that affects PCR results is annealing, so we use a lattice gradient PCR instrument to verify different PCR reaction conditions, mainly the effect of annealing temperature on PCR results, to test the adaptability of this kit to different PCR instruments.

[0078] The freshly collected EDTA anticoagulated blood was prepared according to the method in Example 2 to prepare a PCR reaction solution.

[0079] 20μl reaction system, as shown in Table 7:

[0080] Table 7

[0081] Element

reaction tube

template whole blood

2μl

2×Taq Master Mix

10μl

Primer APOC3-PF / APOC3-P...

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Abstract

The invention discloses a primer for detecting human APOC3 gene mutation. The sequence of a forward primer is APOC3-PF5'-GACCCAGCTAAGGTTCTAC-3' which is shown in SEQ ID No.3. The sequence of a reverse primer is APOC3-PR5'-AGGCTGAAGAGGCACAG-3' which is shown in SEQ ID No.4. In addition, the invention discloses a PCR amplification kit for detecting human APOC3 gene mutation. The kit comprises 2 * Taq Master Mix, the primer pair and deionized water ddH2O. Genome DNA does not need to be extracted, APOC3 genes are directly amplified through a PCR method from blood, after sequencing, APOC3 mutation sites are identified, whole blood can be directly used as a PCR template, detecting cost is reduced, and meanwhile operation pollution is avoided.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a primer and a kit for detecting human APOC3 gene mutation. Background technique [0002] Hypertriglyceridemia (hypertriglyceridemia, HTG) is a kind of hyperlipidemia, which can cause atherosclerosis, cause blood vessel blockage and thrombosis, and in severe cases, it can also cause coronary atherosclerotic heart disease (coronary atherosclerotic heart disease) ,CHD). However, in the mild and moderate stages, the symptoms of high triglycerides are not obvious, and the patient may not feel it at all. When the symptoms of high triglycerides gradually appear, it has developed to the narrowing of blood vessels, affecting the body's blood supply and body. The internal blood supply is insufficient, and the condition has reached a serious level at this time. Once a person is detected as a patient with hypertriglyceridemia, he must change his lifestyle, refuse high-oil, high-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 谢彩霞蔡丽君罗鹏沈伟强
Owner 浙江绍兴鼎晶生物医药科技股份有限公司
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