Mutant detection method and application thereof
A detection method, mutant technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as expensive equipment, difficult to distinguish, time-consuming and labor-intensive
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Embodiment 1
[0077] This example provides a detection method for detecting mutants. In this example, two fluorescent probes are simultaneously labeled in the PCR amplification region. Probe I is labeled with FAM, which can be combined with the target gene to detect the target site Whether the point is mutated, probe Ⅱ uses HEX markers, and the gene in the conserved region near the mutation target is used as the internal reference gene. Probe Ⅱ can be combined with the internal reference gene to evaluate the number of alleles in the overall sample, so as to distinguish Mutate individuals and detect mutation frequencies.
[0078] This embodiment also includes a pair of amplification primers, so that the binding regions of probe I and probe II are all within the amplification range of the primers. like Figure 1-Figure 3 As shown, the newly introduced mutation will destroy the combination of probe I and the amplified fragment but will not affect the combination of probe II, so it is easy to ...
Embodiment 2
[0092] Example 2 Detection of rice homozygous mutants induced by CRISPR-CAS9
[0093] The method provided in Example 1 is used to detect transgenic rice lines (such as Figure 10 shown).
[0094] The 9 families have previously identified 9 independent rice families with mutation patterns by sequencing methods, and their mutation patterns include substitutions, insertions, and deletions. As shown in table 2:
[0095] Table 2 Mutation patterns of rice wild type and TGW6 gene mutants
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[0098] The underlined part of the wild-type sequence in the above table indicates the binding site with the TGW6-HEX probe, and the bold part indicates the binding site of the TGW6-FAM probe; the mutation method in the above table is an example of sample 10-7, -3 Indicates that the mutated mutant is missing 3 bases compared with the wild type, and the brackets indicate that the mutant has 2 bases added after the 5 base deletion compared with the wild type, so the mutant 1...
Embodiment 3
[0100] Example 3 Detection of Rice Heterozygous Mutations Induced by CRISPR-CAS9
[0101] Transgenic rice lines of heterozygous mutants at the TGW6 gene editing site were detected using the method provided in Example 1. Select 3 rice heterozygous individuals with different mutation types including 19-6, (del1, missing 5 bases GCCGC), having the sequence shown in SEQ ID NO.20; 28-4 (del2, missing 4 bases GCCG, A and C replacement, T and G replacement), has the sequence shown in SEQID NO.22; 31-8 (single base deletion), has the sequence shown in SEQ ID NO.27; Real-time quantitative PCR is further passed 2 -△△CT Method analysis The TGW6-HEX probe was used as an internal reference gene to estimate the number of alleles in the overall sample and the wild-type template was used as a calibration sample. Since heterozygous mutants have half the amount of wild-type template and half the amount of mutant template, the relative value should be around 0.5. The results are also as expec...
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