| [Purpose] The determination of the methylation level of the target gene is the basis of biological and medical research involving DNA methylation.The method of using the average methylation level of multiple CpG sites to represent the methylation level of the entire gene is much more accurate than using only one CpG site.So far,There are several methods that can be used to detect the average methylation level of several CpG sites,but these methods are all cost,time-consuming(up to several weeks),and labor-intensive.The most important thing is that these methods cannot complete the measurement of the average methylation level at multiple sites and is difficult to be applied to the early detection of endometrial cancer.Therefore,based on the unique thermodynamic properties of the DNA strand migration process,we constructed a DNA fluorescent probe and used it to determine the average methylation level of multiple CpG sites.The theoretical calculation of the reaction process confirms the basic principle of our probe.Targeting the two CpG sites in the promoter region of the cancer suppressor genes SF-1 and VDR associated with endometrial cancer,we artificially synthesized target sequences with different numbers of mismatches and detected their average methylation with probes Level.Finally,we observe the experimental results to prove the practicability of the probe.[Methods] We first need to consult the relevant literature to select genes related to endometrial cancer and design corresponding probes.Then we artificially synthesize target sequences with different numbers of mismatches(in order to save costs,the mismatch is used to represent methylation in the method principle verification stage),and the average methylation level is detected using a designed probe.Based on the obtained experimental data,we finally establish a standard curve of average methylation level and fluorescence intensity,evaluate the sensitivity of the method,and optimize the probe structure and reaction conditions based on the results.[Results] In the detection of the fluorescence intensity of the 2-3 CpG loci,the fluorescence intensity and the conversion rate of the chain migration probes are arranged in the same difference with the number of mismatches.That is,as the number of mismatched sites increases,the proportion of fluorescent signals decreases.At the same time,the fluorescent signals produced by different synthetic strands with the same number of mismatch sites are almost the same.We mixed the synthetic chain and configured a series of mixed samples.With a precision of 10%,as the average mismatch ratio increased,the fluorescence signal decreased linearly.Moreover,the fluorescence signals produced by different DNA mixed samples with the same mismatch ratio are almost equal.[Conclusions] The novel chain migration probe system we designed can in principle detect the average methylation level of 2-3 CpG sites in a DNA region.Compared with other current detection methods,our method is fast,and the whole process only takes about 2hours.Moreover,our testing costs are low and the instruments are common.Therefore,our method is more suitable for generalization in clinical applications.This can provide a good tool for the detection of average methylation levels at different sites in the future.[Purpose] The first part of the experiment was performed on the synthetic strand.But when we detcct actual clinical samples,the target is genomic DNA.To verify the usability of this method at the genome level,we designed related experiments at the genome level to detect methylation levels.In order to develop new methylation sites as diagnostic markers for endometrial cancer,two CpG sites in the promoter region of the cancer suppressor genes SF-1 and VDR were targeted to measure their average methylation levels in endometrial cancer patients and healthy people,respectively.[Method] We first need to prepare genomic DNA with methylation levels of 0% and 100%at the two CpG sites.According to literature,genomic DNA in human placenta tissue has a very low methylation level,which can be used as a standard sample representing a methylation level of 0%.We then obtained placental tissue from Wuhan Union Hospital and extracted genomic DNA from it as our standard sample.Then,genomic DNA extracted from placental tissue was treated with methylase(M.Sss I)to obtain genomic DNAs as a standard sample representing a methylation level of 100%.Through agarose gel electrophoresis and first generation sequencing,we have proved that we have successfully prepared standard sample representing a methylation level of 0% and 100%.Then we used the standard samples to prepare a serial samples with average methylation levels of 10%,20%,30%,40%,and 50%,and we proved the above samples were successfully prepared by sequencing.Then we amplified the above samples by PCR and used probes to detect the products,and then established a standard curve of average methylation level and fluorescence value.Compared with the results of pyrosequencing,the effect of this method on the average methylation level of target regions of genomic DNA was evaluated.Finally,we applied our method to clinical cancer detection.We collected specimens of endometrial cancer and normal endometrial tissue,and used the method of the present invention to measure the average methylation levels of SF1 and VDR promoter regions of endometrial cancer-related genes.The results were compared with clinical conditions to establish correlations and evaluate the clinical significance of endometrial cancer-related genes as endometrial cancer biomarkers.[Results] At the level of genomic DNA,we used probes to detect samples with different methylation ratios.The results show that with the increase of the average methylation level of DNA,the fluorescence signal increases almost linearly,which proves that our method can detect the average methylation level of two CpG sites in genomic DNA extracted from real clinical samples with a precision of 10%.Finally,when we havedetected the DNA samples of endometrial cancer tissue and normal endometrial tissue,the results show that the fluorescence intensity of DNA produced by cancer patients is higher than that of normal people.The results were in agreement with the inhibitory functions of SF-1 and VDR genes,thus confirming the practicality of the probe.[Conclusion] The methylation level measurement method we designed still has good utility value at the genome level.At the same time,VDR gene as a methylation level measurement site is expected to become a new diagnostic marker for early detection of endometrial cancer. |
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