Novel determination method and reagent for human POLE gene mutation
A mutation and gene technology, applied in the field of diagnostics, can solve the problems of many detection steps, complicated detection steps, and high requirements of operators.
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Embodiment 1
[0094] Example 1. Selection of primers
[0095] The inventors found in experiments that the detection system is very complicated due to the need to simultaneously detect multiple mutants of the POLE gene at one time, resulting in difficult to ensure detection accuracy, large background values, and difficult to overcome the problems of false negatives and false positives.
[0096] In order to find a detection reagent with relatively ideal detection effect, the inventors conducted in-depth research and experimental work based on the characteristics of the POLE gene and its mutation sites, and considered the multi-site / multi-mode primer and probe design strategy, one by one Check the accuracy of the detection (including background interference, false positives and false negatives, etc.).
[0097] After some comparisons, the inventors placed the mutation site at the 3' end of the mutation primer. The inventors have found that the primers are ideal for extension of the mutant temp...
Embodiment 2
[0103] Embodiment 2, the influence of Block primer
[0104] As mentioned above, the present inventors considered the application of Block primers in the design of detection reagents. However, the inventors have found through repeated experiments and analysis that it is not the most appropriate to use Block primers for detection reagents at all sites. In view of the fact that Block primers will increase the complexity of the reaction system, the application of some Block primers will increase the uncertainty of the reaction, or reduce the amplification of mutants, which will have the opposite effect. Therefore, the inventors conducted in-depth analysis of each site, and applied Block primers in the detection reagent for some sites.
[0105] Preparation of reaction templates: Dilute mutant plasmids to 100 copies / μL and wild-type plasmids to 10 5 Copies / μL, respectively, were determined according to the "Fluorescence Quantitative PCR Method" described above.
[0106] For some ...
Embodiment 3
[0112] Embodiment 3, the investigation of sensitivity
[0113]In this example, the inventors respectively mixed different POLE mutant plasmids into pure wild-type plasmids to examine the detection sensitivity.
[0114] Preparation of reaction templates: 100 copies of different mutant plasmids were mixed with 10 5 Copies of wild-type plasmids were mixed; assays were performed as previously described in the "Fluorescence Quantitative PCR Method" and Block primers were applied to some sites as described in Example 2.
[0115] The results are shown in Table 4.
[0116] Table 4
[0117]
[0118] The results showed that 100 copies of the mutant plasmid and 10 5 Copies of wild-type plasmids were mixed and tested as samples with 0.1% mutation abundance. All mutation sites could be detected and could be distinguished from pure wild-type. And all wild-type samples containing mutation sites had Ct greater than 35, and mutant samples had Ct less than 35.
[0119] The above results...
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