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Method for detecting VKORC1 gene polymorphism and nucleic acid probe and kit used in method
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A technology for gene polymorphism and detection probes, used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc.
Inactive Publication Date: 2015-05-06
FUWAI HOSPITAL OF CARDIOVASCULAR DESEASE CHINESE ACAD OF MEDICAL SCI
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At present, CYP2C9 and VKORC1 gene detection has been commercialized, but domestic medical institutions can only use western commercial products for warfarin sensitive gene detection
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Embodiment 1
[0108] 1. Preparation of probes and primer pairs for VKORC1 gene
[0109] A probe for the VKORC1 gene and a primer pair for the VKORC1 gene were prepared using conventional techniques.
[0124] Aliquot, 19 μl per tube into 0.1ml PCR reaction tubes / plates, and transfer to the sam...
Embodiment 2
[0138] Steps one to four:
[0139] In the same method and steps as in Example 1, the DNA sample was replaced with a DNA sample expressing a mutant VKORC1, and 1 μl of the mutantDNA sample was added to 19 μl of the PCR reaction solution.
[0140] 5. Results analysis
[0141] VKORC1 appears to be mutant as a result of Figure 2 show. These graphs are analysis graphs showing changes in fluorescence intensity over time, that is, changes in fluorescence intensity as the number of amplification cycles increases. Such as Figure 2 As shown, the fluorescence curve indicates that the VKORC1 gene in the sample is a mutant type.
Embodiment 3
[0143] Steps one to four:
[0144] In the same method and steps as in Example 1, the DNA sample was replaced with a DNA sample expressing wild-type VKORC1, and 1 μl of the wild-type DNA sample was added to 19 μl of the PCR reaction solution.
[0145] 5. Results Analysis
[0146] VKORC1 expresses wild-type results in Figure 3 show. These graphs are analysis graphs showing changes in fluorescence intensity over time, that is, changes in fluorescence intensity as the number of amplification cycles increases. Such as Figure 3 As shown, the fluorescence curve indicates that the VKORC1 gene in the sample is wild type.
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Abstract
The invention discloses a probe for detecting VKORC1 gene polymorphism. A nucleotide sequence of the detecting probe is selected from a sequence I and a sequence 2, and respectively comprises a fluorescent group and a quenching group at 5' end and 3' end; the wavelength peak lengths of fluorescent lights emitted from the fluorescent group are at different positions; preferably, the quenching group is selected from MGB and BHQ2; preferably, the fluorescent group is selected from FAM, VIC, JOE, HEX, CY3, NED, TAMRA, ROX, TEXAS, RED, CY5 and the like. The invention further discloses a primer pair for gene amplification. The primer pair comprises a positive primer and a reverse primer, wherein the positive primer is oligonucleotides formed by a base sequence in a sequence 3; and the reverse primer is oligonucleotides formed by a base sequence in a sequence 4. The invention further discloses a kit containing the primer and the probe, and a method for detecting CYP2C9*2 gene polymorphism.
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