Primer set, kit and method for detecting cardiovascular disease-related gene polymorphisms
A gene polymorphism and cardiovascular technology, which is applied in the field of reagent kits, biological molecular detection, and primer sets for detecting cardiovascular disease-related gene polymorphisms. It can solve the problems of high detection costs, high design costs, and lack of specificity. problems, to achieve high accuracy, low cost, and strong specificity
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Embodiment 1
[0092] Embodiment 1: the design of primer
[0093] Principles and considerations for primer design:
[0094] 1. Length: 15bp to 30bp, its effective length [Ln=2(G+C)+(A+T)] is generally not greater than 38, otherwise the optimum extension temperature of PCR will exceed the optimum action temperature of Taq enzyme, thus Reduce product specificity.
[0095] 2. G+C content: it should be between 40% and 60%. The annealing temperature in PCR amplification is generally the Tm value of the primer with a lower Tm value minus 5 to 10 degrees. When the primer length is less than 20, its Tm is always equal to 4×(G+C)+2×(A+T).
[0096] 3. The randomness of base distribution: more than 4 single bases in a row should be avoided. In particular, there should not be more than 3 consecutive Gs or Cs at the 3' end, otherwise the primers will be misprimed in the G+C-rich sequence region.
[0097] 4. The primer itself: it cannot contain a self-complementary sequence, otherwise a hairpin-like s...
Embodiment 2
[0102] Embodiment 2: Primer sequence screening detection
[0103] (1) Divide into 3 reaction tubes, Pool 1, Pool 2, and Pool 3, and add 10 ng of human genomic DNA (DNA sample of oral epithelial exfoliated cells) to each reaction tube.
[0104] (2) Using the DNA sample added in step (1) as a template, the PCR amplification primers (composed of SEQ ID NO: 1 to SEQ ID NO: 42) obtained in Example 1 were used for multiplex PCR amplification.
[0105] Wherein, the PCR amplification primers were combined in equal proportions (ie 1:1(v)) according to the method in Table 2, oscillated and mixed; and used after brief centrifugation.
[0106] Among them, the PCR amplification reaction system of each of Pool 1, Pool 2, and Pool 3 is: primer premix 10 μL, 2XPCR MIX 12.5 μL, template (positive quality control) 1 μL, ddH 2 Make up 25 μL of O, vortex and mix well, and perform multiple PCR amplification after brief centrifugation. The PCR reaction conditions include: stage 1: 95°C for 3 minut...
Embodiment 3
[0114] Embodiment 3: Primer sequence screening detection
[0115] The difference between this example and Example 2 is that the PCR amplification primers used in step (2) consist of SEQ ID NO:43 to SEQ ID NO:84. The detection result of this embodiment is as follows figure 2 shown.
[0116] From figure 1 and figure 2 The results obtained are shown in Table 4.
[0117] Table 4
[0118]
[0119] Depend on figure 1 and figure 2 As can be seen from Table 4, the primer sets detected in Examples 2 and 3 all obtained the typing information of all loci. Will figure 1 and figure 2 Comparing it can be seen that the peak height consistency between the sites of the primer set detected in Example 3 is better than that of the primer set detected in Example 2, so the primers consisting of SEQ ID NO:43 to SEQ ID NO:84 are recommended The set (set of primers shown in Table 1) is the preferred sequence information.
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