Method for enriching mutant gene sequence and application
A technique for mutating genes and sequences, which is applied in the field of enriching mutated gene sequences, can solve problems such as poor sensitivity, and achieve the effects of increasing sensitivity, increasing copy number, and increasing quantity
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Embodiment 1
[0065] This embodiment provides a method for enriching mutant gene sequences, the flow chart is shown in figure 1 , with the following steps:
[0066] S1: Preparation of hybrid DNA: The plasma free DNA (cfDNA) is directly prepared for the hybrid sequence, and the tissue genome DNA needs to be broken into a sequence of about 200 bp by ultrasound before the hybrid sequence is prepared. The tumor DNA gene detection sequence contains mutant sequences and also contains a large number of wild-type normal sequences. Use high temperature denaturation to change double-stranded DNA into single-stranded DNA, and then anneal the denatured DNA by gradient cooling according to the Surveyor enzyme instructions, and anneal the mutated gene sequence and the corresponding normal gene sequence into a hybrid sequence ( figure 2 ).
[0067] (1) The conditions for double-stranded DNA denaturation and renaturation are: 95°C for 10 minutes, 95°C to 85°C (-2.0°C / s), 85°C for 1 minute, 85°C to 75°C ...
Embodiment 2
[0089] 1) Extract plasma cell-free DNA of lung cancer patients by using Aide Bio-free Nucleic Acid Extraction Kit, take 20ng of cfDNA and directly use it for digital PCR to detect the copy number of mutated genes, and at the same time, 20 ng of cfDNA enriches the mutated gene and then performs digital PCR to detect the mutated gene copy number.
[0090] 1. The detected mutation site is the E746-A750 site mutation in exon 19 of the EGFR gene. The primer and probe information for digital PCR to detect the copy number of the mutant gene is as follows:
[0091] a. Directly use digital PCR to detect the primers and probe information of the mutant gene as follows:
[0092] Probe sequence:
[0093] 5'-VIC-AGGAATTAAGAGAAGCA-MGB-NFQ-3' (SEQ ID NO.7);
[0094] 5'-FAM-CGCTATCAAAACATCTC-MGB-NFQ-3' (SEQ ID NO.8);
[0095] Primer sequence:
[0096] F: 5'-TCCTCTCTGTCATAGGGACTCTG-3' (SEQ ID NO.9);
[0097] R: 5'-GAGGATTTCCTTGTTGGCTTTC-3' (SEQ ID NO.10);
[0098] b. The information of pr...
Embodiment 3
[0123] The method in Example 1 was applied to the detection of mutation genes in tumor patients.
[0124] Samples with a mutation frequency of 1% were mixed with plasma DNA from the normal population for 2-fold gradient dilution. The mutation frequencies of samples before and after dilution were: 1%, 0.5%, 0.25%, 0.125%, 0.0625%, and 0.0313%. , 0.0156%, 0.0078%; take 40ng of samples diluted into different mutation frequencies and add an equal volume of water for 2-fold gradient dilution. The sample volumes before and after dilution are: 40ng, 20ng, 10ng, 5ng, 2.5ng, 1.25ng, 0.625ng, 0.313ng. The diluted samples were enriched according to different DNA template amounts and mutation frequencies. The enriched samples were dissolved in 30 μL double-distilled water and tested by fluorescent quantitative PCR. Each sample was tested by eight independent PCR tubes. , and calculate the detection rate. The detection rate was calculated as the number of detected positive tubes divided ...
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