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Comt site-related primers and polymorphism detection kits for this site

A technology related to primers and kits, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of high cost, inability to type correctly by real-time quantitative PCR, and difficulty in clinical promotion, etc. To achieve the effect of simple operation

Active Publication Date: 2020-02-11
CHANGCHUN HENGXIAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sequencing method requires a relatively complex experimental platform, and requires subsequent processing after sample amplification, which is difficult to promote clinically.
The real-time quantitative PCR method has the advantages of simple operation and no need for post-amplification treatment, but due to the need to set up an internal control, its cost is high
Moreover, about 4 / 1000 people in the population have COMT Val158Met in close proximity to another SNP site, resulting in incorrect typing by real-time quantitative PCR
Although 4 per 1,000 is not a very high ratio, there is a huge loss for each mistyped individual

Method used

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  • Comt site-related primers and polymorphism detection kits for this site
  • Comt site-related primers and polymorphism detection kits for this site
  • Comt site-related primers and polymorphism detection kits for this site

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: the dissolution and detection of four kinds of primers

[0057] (1) Dissolving primers

[0058] Since Oligo DNA is in the form of a light dry film lying on the tube wall, it is easily lost when opening, so please centrifuge first when opening the tube cap, then slowly open the tube cap, add enough water when dissolving, and then cover the tube cap , full shock 5-10min.

[0059] (2) Primer detection

[0060] 1. Detection of primer concentration

[0061] Use an ultraviolet spectrophotometer and pure water to measure the blank value, take 2ul primers (10uM), and measure the nucleic acid concentration.

[0062] The detection results of the four primers should meet the following requirements:

[0063] Forward amplification primer OD （260 / 280） The detection result is 10uM: 70-100ng / ul; reverse amplification primer OD （260 / 280） The detection result is 10uM: 70-100ng / ul; G extension primer OD （260 / 280） The detection result is 10uM: 40-60ng / ul; A extension p...

Embodiment 2

[0066] Embodiment 2: detection kit and its use

[0067] (1) Prepare a kit including the following components: 1 tube of primer mixture (36 μl / tube), 1 tube of primer mix ② (36 μl / tube), 1 tube of PCR reaction solution (420 μl / tube), 1 tube of deionized water (500 μl / tube), 1 tube each of positive and negative controls (10 μl / tube).

[0068] (2) Specimen collection, transportation and storage:

[0069] 1. Specimen collection: The specimens are blood, amniotic fluid, and villous tissue. The blood is 2ml of venous blood or 0.5-1ml of fetal umbilical cord blood, which is anticoagulated with EDTA; 2-5ml of amniotic fluid or two pieces of villi tissue are obtained by puncture.

[0070] 2. Storage: It can be detected immediately, stored at 4°C for one week, and stored at -20°C for up to one year.

[0071] 3. Transportation: Specimens should be transported using 0°C ice cubes.

[0072] (3) Detection steps and result analysis:

[0073] 1. DNA extraction

[0074] The Qiagen DNA...

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Abstract

The invention relates to primers related to a COMT site, belonging to the field of biotechnology. The objective of the invention is to design four primers so as to obtain the primers with sequencing accuracy related to the COMT site and a detection kit for the gene polymorphism of the COMT site. The primers comprise a forward amplification primer, a reverse amplification primer, a G extension primer and an A extension primer. The invention provides a specific primer extension method which has sequencing accuracy, does not misjudge any genetic typing and has the advantage of simple real-time quantitative PCR operation.

Description

technical field [0001] The invention belongs to the field of biotechnology. Background technique [0002] Parkinson's disease, also known as idiopathic Parkinson's disease (PD), referred to as Parkinson's disease, is one of the common neurodegenerative diseases in middle-aged and elderly people. The prevalence rate of people over 65 years old is about 2%, increasing with age, more men than women. The main clinical features of the disease: resting tremor, slowness and decrease in movement, increased muscle tone, and unstable posture are the main features. [0003] Parkinson's disease should emphasize comprehensive treatment, including drugs, physical therapy, hydrotherapy, medical sports, daily life adjustment and surgery, etc., rather than a single treatment method. Drugs for the treatment of Parkinson's disease have been developed to the third generation. First-generation anticholinergics; second-generation levodopa; third-generation dopamine receptor agonists and enhanc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2535/125C12Q2531/113C12Q2563/107
Inventor 杜培革蔡燕宁于顺安利萍王敖雪赵雪隋宝珍王玉华关恒
Owner CHANGCHUN HENGXIAO BIOTECH CO LTD