Method for detecting ApoE single nucleotide polymorphism
A single nucleotide polymorphism, to-be-detected technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as time-consuming, cumbersome operation steps, error-prone, etc. Clear purpose, simple data analysis, clear targeting effect
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Embodiment 1
[0042] Example 1. Establishment of a method for detecting ApoE single nucleotide polymorphisms
[0043] The method for detecting ApoE single nucleotide polymorphism comprises the steps:
[0044] 1. Target gene sequence information
[0045] Schematic diagram of the distribution of ApoE alleles figure 1 As shown, the ApoE coding gene is shown in SEQ ID NO.23. There are two SNP sites at the 241bp and 379bp respectively, and there is a T>C variation at 241bp, that is, rs429358 (c.388T> C), its wild type is T, and its mutant type is C; there is a C>T variation at 379bp, that is, rs7412 (c.526C>T), its wild type is C, and its mutant type is T; thus the present invention Primer and probe sequences were designed and detected for the following four genotypes in the ApoE coding gene:
[0046] (1) ApoE gene SNP site rs429358(c.388T>C)T genotype and rs7412(c.526C>T)C genotype;
[0047] (2) ApoE gene SNP site rs429358(c.388T>C)T genotype and rs7412(c.526C>T)T genotype;
[0048] (3) Ap...
Embodiment 2
[0100]Embodiment 2.A1, A2, A3, the detection of A4 system
[0101] Prepare according to the system requirements in Table 3 and Table 4, and the sample volume of the template is 10 6 copy / uL, get the results in the accompanying drawings respectively figure 2 -a1, figure 2 -a2, figure 2 -a3, figure 2 -a4, design primers upstream and downstream of the SNP sites of rs429358 and rs7412, which are used for upstream and downstream primers of fluorescent quantitative PCR detection. The 5' end of the probe sequence is labeled with a fluorescent group, and the 3' end of the probe sequence is labeled with a quencher group. The position of the mutated base is different, and the specificity that appears is also different. When the probe used When the SNP mutation site is at the 5th position of the 5' end (P1, P2, P3, P4), the fluorescence detection result is low and cannot be typed. When the SNP mutation site of the probe used is at the first position of the 3' end (P5, P6, P7, P8...
Embodiment 3
[0105] The detection of embodiment 3.A5 system and A6 system
[0106] According to the A5 system and A6 system, the product was prepared for fluorescence detection, and the sample volume of the template was 10 6 copies / uL, Pic 4-1 The result is the test result of A6 system, Figure 4-2 The results are the detection results of the A5 system, and the results show that adding a set of mutation primers at SNP positions can greatly increase the typing ability of the product (A6 system).
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