Establishment Of DNA Methylation Detection Method And Its Application In Serum Samples From Breast Cancer

The high incidence rate of breast cancer has seriously affected the physical and mental health of women in our country.Early detection and treatment can reduce the mortality rate of breast cancer.Researches show that whether the promoter region of the gene is methylated or not can be an important biological indicator for early screening breast cancer.With the rapid development of"liquid biopsy"technology,it has important clinical significance for early diagnosis of the tumor to monitor the methylation level of cell-free DNA.Therefore,the paper established an integration method for assessing DNA methylation in serum samples from breast cancer patients to provide new ideas for early screening of breast cancer.1. A nucleic acid extraction kit based on magnetic bead method was optimized for serum samples,including the number of magnetic beads and acryl carrier.And a series of performance tests were carried out on the kit,such as stability test and recovery test.The results showed that the kit had stability greatly and its recovery rate of nucleic acid was over 90%.Meanwhile,it had the same or even better extraction effect on clinical samples than the commercial column method kit.The extraction time of the kit also was shortened through optimizing the extraction steps,integrating the lysis and binding steps.2. A new method that RAA fluorescence detection for detecting methylation of SNRPN gene was developed.The experiment includes that primers and probes for RAA fluorescence detection were synthesized and screened.Meanwhile,the reaction system was optimized,such as reaction time and reaction temperature.Finally,the standard methylation template was synthesized for drawing the quantitative curves of methylated genes.The results showed that there is a correlation when the concentration of the methylated template was 4.64×10~8 copies/?L-4.64×10~4 copies/?L.Therefore,the method can quantitatively evaluate the methylation of SNRPN gene.3. The RASSF1A gene by using the nucleic acid extraction kit and the RAA fluorescence methylation detection was detected in serum samples from breast cancer patients.25 breast cancer serum samples were analyzed.First the obtained nucleic acids after treated were divided into two groups,one was detected by MSP method and the other by RAA fluorescence detection method.The detection results were as follows:the detection rate of MSP method was 52%(13/25),and that of RAA fluorescence method was 56%(14/25).The difference between the two groups was caused,due to the low template concentration in one sample.Consequently,the results showed that the RAA fluorescence detection method meets the requirements of methylated gene screening and has higher sensitivity than the MSP method.