Fluorescent quantitative kit for fast detecting SNP loci
A fluorescence quantitative and kit technology, applied in the field of molecular biology, can solve the problems of cross-contamination, low selectivity and low sensitivity of SNP site detection, and achieve the effects of short time, saving time and labor costs, and high sensitivity
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[0025] Example 1: Amplification and detection of gene Kras mutation site rs121913529
[0026] The mutant Kras plasmid DNA Mutant Target containing the rs121913529 site and the wild-type Kras plasmid DNA Wild Target containing the rs121913529 site are respectively used as detection targets to verify the sensitivity and specificity of the present invention. The Kras plasmid DNA sequence is shown in Table 1 (shown DNA sequence and its complementary strand).
[0027] Table 1 Plasmid sequence
[0028]
[0029]
[0030] The primers in the kit are designed according to the Kras mutation site rs121913529, including the first type primer DP1, the second type primer P2, the third type primers P3 and P4, and the rU in the first type primer DP1 is ribonucleotide, and Kras The target mutation site in the sequence is complementary, and C3Spacer is modified at the 3'end to block the extension reaction of the first primer DP1 by the nucleic acid polymerase. The corresponding sequence and end modifi...
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[0043] Example 2: Amplification and detection of rs121913529 mutation site in cell genomic DNA
[0044] 10 respectively 6 HT29 cells (rs121913529 mutation negative) and SW480 cells (rs121913529 mutation positive) were extracted with ONE-4-ALL Genomic DNA Mini-Preps Kit, and then 1% or 0.1% of the cell extract was taken, and the kit of the present invention was used to perform For the amplification and detection of the rs121913529 mutation site, the amplification and detection mixture used is the same as in Example 1. Please refer to the attachment for the real-time fluorescence curve of the analysis result Figure 4 . The real-time fluorescence curve shows that two different concentrations of rs121913529 mutation-positive cells SW480 gave real-time fluorescence curves with different POI values around 40 minutes, and different concentrations of rs121913529 mutation-negative cells HT29 did not give real-time fluorescence curves within 90 minutes. It shows that the kit of the pr...
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