Primer group and kit for simultaneously detecting multiple mutations of nine genes related to congenital adrenal hyperplasia
A technology of adrenal cortex and primer set, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve missed detection and false detection, difficulty in distinguishing true and false genes, and uncertainty whether mutations are linked, etc. problem, to achieve the effect of wide detection range, detection false detection and low detection rate
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Embodiment 1
[0105] Embodiment 1: Utilize the multiplex PCR method of the present invention to amplify CAH-associated gene mutation
[0106] Prepare the reaction system according to Table 9 below to amplify peripheral blood, dried blood spots and genomic DNA samples.
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[0108] On the PCR instrument, perform pre-amplification according to the conditions shown in Table 10 below:
[0109]
[0110] After the amplification is completed, take 5 ul of each sample and detect it on a 1% DNA gel. The results are as follows: figure 2 As shown, using different samples as templates, CAH-related genes can be effectively amplified.
Embodiment 2
[0111] Example 2: Construction of a PacBio sequencing library using the multiplex PCR method involved in the present invention
[0112] Step 1: Multiplex PCR Amplification
[0113] Prepare the reaction system according to the following table 11, and amplify the peripheral blood samples of different types of CAH-related gene mutations:
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[0115] On the PCR instrument, perform pre-amplification according to the conditions shown in Table 12 below:
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[0117] After the amplification is completed, put the amplification product into a centrifuge, centrifuge at 10000rpm for 20min. After the centrifugation, place it horizontally, take 4 μL of the supernatant and add it to a new tube.
[0118] Step 2: Construct the PacBio sequencing library
[0119] Prepare reaction system according to following table 13:
[0120]
[0121] On the PCR instrument, react according to the following conditions: 37ºC for 20 minutes; 25ºC for 15 minutes; 65ºC for 10 minutes. After...
Embodiment 3
[0124] Example 3: Detection and verification of CAH-related gene mutations
[0125] The heel blood of 14 subjects and the peripheral blood genomic DNA of 11 subjects were collected from Hunan Jiahui Genetic Specialized Hospital as 25 cases of verification samples. Referring to Example 2, the primer set (and kit) of the present invention was used to simultaneously Multiple mutations in 9 gene loci related to CAH were detected. At the same time, the copy number of CYP21A2 gene was detected by MLPA method, and the point mutation of related genes was detected by Sanger sequencing. The results obtained by using the present invention are compared with the control results, the results are shown in Table 14, and the results of 25 samples are completely consistent.
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[0129] Therefore, the specificity and sensitivity of the results detected by the method of the present invention are 100% compared with those of MLPA combined with PCR-Sanger sequencing...
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