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目的探讨Ras相关区域家族1A(RASSF1A)基因作为通用胎儿DNA标记的可行性,建立Real-time PCR为基础的非侵袭性产前胎儿RHD基因型定型平台。方法抽取未孕自愿者7名(男2名,女5名)和43例Rh D阴性孕妇的EDTA抗凝血5 ml,分离血浆抽提其中的游离细胞DNA。联合使用5种甲基化敏感性限制性内切酶对未/低甲基化的母源性RASSF1A基因进行酶切以保留高度甲基化的胎源性RASSF1A基因;同时酶切未甲基化的β-actin基因作为对照,以检测酶切的彻底性;通过Real-time PCR扩增酶切前的RASSF1A基因和β-actin基因、酶切后的RASSF1A基因,分别检测血浆总游离细胞DNA和胎儿游离细胞DNA;对确认存在胎儿DNA的标本,扩增RHD基因的外显子5和7,以检定胎儿的RHD基因型。结果 7例未孕自愿者和43例孕妇酶切处理前的RASSF1A基因和β-actin基因扩增均阳性。酶切处理后,7例未孕自愿者和2例孕妇未检测到RASSF1A基因和β-actin基因,41例孕妇检测到RASSF1A基因、未检出β-actin基因,其中39例检出RHD基因外显子5和7。结论RASSF1A基因可作为通用的胎儿DNA标记,荧光定量PCR扩增RHD基因外显子5和7的平台可用于非侵袭性胎儿RHD基因型别检定。
Objective To investigate the feasibility of RASSF1A gene as a universal marker of fetal DNA and to establish a real-time PCR-based non-invasive prenatal fetal RHD genotyping platform. Methods A total of 7 volunteers (2 males and 5 females) and 5 pregnant women (43 Rh D-negative pregnant women) were enrolled in this study. Blood samples were collected for free DNA extraction. Unmethylated / hypomethylated maternally derived RASSF1A gene was digested with 5 methylation-sensitive restriction enzymes to retain the highly methylated fetal-derived RASSF1A gene; digestion of unmethylated Β-actin gene as a control to detect the thoroughness of the enzyme digestion. The RASSF1A and β-actin genes before digestion and the RASSF1A gene after digestion were amplified by Real-time PCR, and the total free plasma DNA Fetal free cell DNA; To confirm the presence of fetal DNA samples, amplification of RHD gene exons 5 and 7, to test the fetus RHD genotype. Results The RASSF1A gene and β-actin gene were positive in 7 unrelated volunteers and 43 pregnant women before digestion. RASSF1A gene and β-actin gene were not detected in 7 cases of un-pregnant volunteers and 2 cases of pregnant women. RASSF1A gene was detected in 41 pregnant women and β-actin gene was not detected. Of 39 cases, RHD gene was detected Exons 5 and 7. Conclusion The RASSF1A gene can be used as a general fetal DNA marker. Fluorescent quantitative PCR can be used to amplify RHD exon 5 and 7 in non-invasive fetal RHD genotypes.