Composition for detecting single nucleotide polymorphism an application of composition
A single nucleotide polymorphism and composition technology, which is applied in the field of gene fluorescent probe detection, can solve the problems of time-consuming, time-consuming, labor-intensive, and difficult operations, and achieves a reduction in the probability of non-specific binding, design difficulty, and development savings. cost effect
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Embodiment 1
[0043] Embodiment 1: as figure 1 As shown, the allele-specific primers and their associated probes were designed and used for the detection of the rs429358 (T / C) SNP site of the ApoE gene; the PCR template was the genomic DNA of the cell line.
[0044] Reaction liquid system preparation: H 2 O, purified water, 16.7 μL; PCR buffer (Mg 2+ plus), 5X, 5μL; dNTPs (each 10mM dATP, dCTP, dGTP; 20mM dUTP), 10mM, 0.5μL; allele-specific primer 1, 100uM, 0.1μL; allele-specific primer 2, 100uM, 0.1μL; Allele-specific primer-associated probe 1, 100uM, 0.05μL; Allele-specific primer-associated probe 2, 100uM, 0.05μL; Upstream primer, 100uM, 0.1μL; Downstream primer, 100uM, 0.1μL; UDG enzyme, 1U / μL, 0.1μL; polymerase, 5U / μL, 0.2μL; total volume 23μL.
[0045] Operation steps: Prepare relevant liquids according to the above reaction liquid system preparation, shake and mix for 15 seconds, and centrifuge quickly for 15 seconds, dispense 23 μL per tube into PCR reaction tubes, add 2 μL of ...
Embodiment 2
[0059] Embodiment 2: as figure 2 As shown, the allele-specific primer complementary probe PCR detection technology was used for the detection of the MTHFR gene 677 (C / T) SNP site; the PCR template was the genomic DNA of the cell line.
[0060] Reaction liquid system preparation: H 2 O, purified water, 16.7 μL; PCR buffer (Mg 2+ plus), 5X, 5μL; dNTPs (each 10mM dATP, dCTP, dGTP; 20mM dUTP), 10mM, 0.5μL; allele-specific primer 1, 100uM, 0.1μL; allele-specific primer 2, 100uM, 0.1μL; Allele-specific primer-associated probe 1, 100uM, 0.05μL; Allele-specific primer-associated probe 2, 100uM, 0.05μL; Upstream primer, 100uM, 0.1μL; Downstream primer, 100uM, 0.1μL; UDG, 1U / μL, 0.1 μL; polymerase, 5 U / μL, 0.2 μL; total volume 23 μL.
[0061] Operation steps: Prepare relevant liquids according to the above reaction liquid system preparation, shake and mix for 15 seconds, and centrifuge quickly for 15 seconds, dispense 23 μL per tube into PCR reaction tubes, add 2 μL of the samples...
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