Primer for detecting K-ras genic mutation and application thereof
A genome and primer sequence technology, which is applied in the field of primers for detection of K-ras gene codon 12/13 and 61 mutations, can solve problems such as not widely developed and immature technical conditions, and achieve high reference significance
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Embodiment 1
[0029] Example 1 A method for identifying K-ras codon 12 / 13 mutations
[0030] 1. Extraction of genomic DNA from tumor patient tissues;
[0031] 2. Perform PCR amplification. The 20 μl PCR reaction system is as follows: 50-100 ng of genomic DNA of the tumor tissue to be tested, 0.125 μl of Taq enzyme, 1 μl of upstream and downstream primers (10 μM), 2 μl of dNTP (2.5 mM), 10 × PCR buffer ( Contains Mg 2+ ) 2 μl, and the rest was sterile distilled water; the PCR reaction conditions were: denaturation at 95°C for 2 min, followed by denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min, and 45 cycles, and finally extension at 72°C for 7 min, and storage at 4°C;
[0032] 3. Gel electrophoresis analysis of PCR amplification products:
[0033] a. Rinse the electrophoresis tank and comb with distilled water. Put it on a level table and set up the comb. 1.5% agarose can be prepared for electrophoresis.
[0034] b. Put 50ml of 1×TAE electropho...
Embodiment 2
[0053] Example 2 A method for identifying the mutation of K-ras codon 61
[0054] 1. Extraction of genomic DNA from tumor patient tissues;
[0055] 2. Perform PCR amplification. The 20 μl PCR reaction system is as follows: 50-100 ng of genomic DNA of the tumor tissue to be tested, 0.125 μl of Taq enzyme, 1 μl of upstream and downstream primers (10 μM), 2 μl of dNTP (2.5 mM), 10 × PCR buffer ( Contains Mg 2+ ) 2 μl, the rest was sterile distilled water; the PCR reaction conditions were: denaturation at 95°C for 2 minutes, followed by denaturation at 95°C for 30 sec, annealing at 50°C for 30 sec, extension at 72°C for 1 min, and 45 cycles, and finally extension at 72°C for 7 min, and storage at 4°C;
[0056] 3. Gel electrophoresis analysis of the PCR amplification product: the specific steps are the same as in Example 1.
[0057] The result is as figure 1 As shown, band 1 represents the PCR product of K-ras codon 61 primer 1, and the product is 426 bp; Band 2 represents the ...
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