High-specificity kit for detecting deafness predisposing genes and uses
A technology of deaf susceptibility genes and detection kits, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of many false negatives and false positives, difficult interpretation of results, and poor repeatability, so as to prevent false positives. Effects of positive and false negative, increasing Tm value, shortening primer length
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Embodiment 1
[0069] The kit of the present invention detects mutations and DNA samples of normal individuals. The primers used for the detection of mutation hotspots 2168A>G, IVS7-2A>G, 1174A>T, 1226G>A, 1975G>C, 2027T>A of the deaf susceptibility gene SLC26A4 were labeled with blue fluorescent dyes, GJB2 176del16bp, 235delC, The primers for 299delAT detection were labeled with green fluorescent dye, the primers for Amelogenin locus and 12S rRNA 1494C>T, 1555A>G mutation detection were labeled with yellow fluorescent dye, and the internal standard was labeled with red fluorescent dye.
[0070] 1. The 1,000 samples to be tested have all been sequenced and detected by using the technical method of "DNA extraction-PCR amplification-sequencing". Among them, 10 samples of IVS7-2A>G mutation, 10 samples of GJB2 235delC mutation, 10 samples of 12SrRNA 1555A>G mutation, 1 sample of 1494C>T mutation, 1 sample of 2168A>G mutation, 1 sample of 1174A>T mutation 1 sample of 1226G>A mutation, 1 sample ...
Embodiment 2
[0096] The unmodified primers were used to detect DNA samples from normal individual blood spots at the same concentration (5.0ng / 25uL system). The labeled primers are the same as in Example 1. The non-labeled primers used for the detection of deaf disease susceptibility loci were the common primers corresponding to the primers in Example 1 without LNA modification.
[0097] 1. The sample to be tested is the same as the normal individual blood spot in Example 1.
[0098] 2. Genomic DNA extraction from samples
[0099] Genomic DNA was extracted by Chelex method.
[0100] 3. Detection and analysis of amplification and amplification products
[0101] 3.1PCR amplification system:
[0102]
[0103] 3.2 PCR amplification procedure: Same as Example 1.
[0104] 4. Fluorescent detection of the amplified product on a genetic analyzer
[0105] With embodiment 1.
[0106] 5 Conclusion
[0107] The result is as Figure 13 As shown, for male normal individuals, the unmodified pr...
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