Primer and kit for detecting human low oxygen tolerance related gene polypeptide
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A gene polymorphism and kit technology, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. And other issues
Pending Publication Date: 2019-08-27
NANTONG UNIVERSITY
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[0007] In order to solve the technical problems in the detection of EGLN1 gene polymorphism related to human hypoxia tolerance by the above method, the detection result is not accurate, the detection cycle is long, the operation is cumbersome, and it is difficult to meet the requirements of clinical testing. The present invention provides a detection result A primer pair and kit for detecting gene polymorphisms of EGLN1 RS479200 and EGLN1 RS480902 related to human hypoxia tolerance, which are accurate, specific, short-term, low-cost, quantitative, and effectively meet the requirements of clinical testing; phospho-sequencing
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Embodiment 1
[0059] Embodiment 1: the preparation of kit (30 tests / box)
[0060] 1. Design and synthesis of primers and probes
[0061]For the polymorphic sites EGLN1 RS479200 and EGLN1RS480902 detected in the human EGLN1 gene, specific mutation sites were selected, and primers were designed using PyroMark Assay Design2.0 software; the forward amplification primer, reverse amplification primer and sequencing primer were first Purified by PAGE and then purified by HPLC, wherein the 5' of the reverse amplification primers of EGLN1 RS479200 and EGLN1 RS480902 are labeled with biotin.
[0062] Table 1. Mutation site and type
[0063] mutation site base change EGLN1 (RS479200) C>T EGLN1 (RS480902) C>T
[0064] The amplified sequence is shown in Table 2:
[0065] Table 2. Specific amplification primers and primer sequences
[0066]
[0067]
[0068] 2. Composition of PCR reaction solution
[0069] Table 3. Composition of PCR reaction solution 1
[0070] ...
Embodiment 2
[0088] Embodiment 2: the use of kit
[0089] 1. Sample testing
[0090] Dissolve the dry powder of the primers (the validity period of the primers is 1 month after dissolution). Prepare the system according to the number of templates: take the PCR reaction solution, add dissolved primers, uracil DNA glycosylase, and Taq DNA polymerase, aliquot the system, add sample DNA, blank control substance or positive control substance as templates, and form PCR reaction system. Perform PCR amplification according to the PCR reaction procedure.
[0091] The main components of the EGLN1 RS479200 and EGLN1 RS480902 systems are as follows:
[0092] Table 9. Main components of EGLN1 RS479200 system
[0093]
[0094] Table 10. Main components of EGLN1 RS480902 system
[0095]
[0096] The system reaction procedure is as follows:
[0097] Table 11. PCR reaction program
[0098]
[0099]
[0100] After the amplification is completed, check the PCR results on agarose gel to pro...
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Abstract
The invention relates to a primer and a kit for detecting human low oxygen tolerance related gene polypeptide, and belongs to the technical field of in vitro nucleic acid detection. A primer group includes a forward amplification primer, a reverse amplification primer and a sequencing primer. A 5'-end is added with a biotin label. The kit includes a forward amplification primer, PCR reaction liquid containing a reverse amplification primer, a sequencing primer, uracil DNA glycosylase, Taq polymerase and a positive control. The kit provided by the invention has the advantages of accurate detection results, high specificity, short detection period, simple operation and effectively satisfying clinical testing requirements. In addition, the kit also has the advantages of real-time monitoring on reaction progress and short reaction time, can perform direct sequencing with a pyrophosphoric acid sequencing machine through simple treatment on PCR products, has higher sensitivity than a gold standard method, namely a capillary electrophoresis sequencing method, and is more suitable for mutation analysis.
Description
technical field [0001] The invention relates to the technical field of in vitro nucleic acid detection, in particular to a primer pair and a kit for detecting gene polymorphisms related to human hypoxia tolerance. Background technique [0002] With the development of the west and the rise of tourism, the exchanges between the plains and plateaus are becoming more and more frequent, and more and more people enter the plateaus from the plains. However, when humans enter the plateau environment from the plains, they are faced with various challenges such as low pressure, low oxygen, low temperature and strong ultraviolet rays, and are prone to acute high-altitude disease (AHAD, acute high-altitude disease). Many people will also be affected by Acute Mild Mountain Sickness (AMS, acute mountain sickness), a syndrome of headaches, difficulty falling asleep, dizziness / dizziness, gastrointestinal symptoms, weakness / fatigue, and some people will even suffer from Acute severe altitud...
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