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目的探讨利用母体血浆中高甲基化RASSF1A位点进行胎儿SNP分型的应用价值。方法随机收集10个未孕健康妇女和45例不同孕期(早期5例、中期20例、晚期20例)孕妇的血样本及相应胎儿组织(绒毛组织、羊水、胎盘组织);利用甲基化敏感限制性内切酶BstUI酶切后进行PCR,产物进行血细胞、血浆和胎儿组织(绒毛或胎盘)DNA RASSF1A序列的甲基化模式检测,并采用直接测序法对SNP rs4688725位点进行分型。结果经BstUI酶消化,RASSF1A序列在母体血细胞中均未检出,而在绒毛或胎盘组织中均能检出;在45名孕妇血浆中,RASSF1A序列均能被检出,且序列内的SNP分型与相应胎儿组织一致;在10名非孕妇女血浆中均未检出RASSF1A序列。结论母体和胎儿DNA中RASSF1A基因启动子区域的甲基化模式存在差异,可用于对母体血浆中的游离胎儿DNA进行SNP分型。
Objective To investigate the value of fetal SNP typing using the hypermethylated RASSF1A locus in maternal plasma. Methods Blood samples and corresponding fetal tissues (villi, amniotic fluid and placenta) from 10 non-pregnant healthy women and 45 pregnant women with different pregnancy stages (5 early stage, 20 middle stage and 20 late stage) were collected randomly. The methylation-sensitive Restriction endonuclease BstUI digestion PCR, the product of blood cells, plasma and fetal tissue (villi or placenta) DNA RASSF1A methylation pattern detection, and direct sequencing of the SNP rs4688725 site typing. Results The RASSF1A sequence was not detected in maternal blood cells but was detected in the villus or placenta tissue by the BstUI enzyme digestion. The RASSF1A sequence was detected in the plasma of 45 pregnant women, and the SNP score Type consistent with the corresponding fetal tissue; RASSF1A sequence was not detected in the plasma of 10 non-pregnant women. Conclusion The methylation patterns of RASSF1A promoter region in maternal and fetal DNA are different, which can be used for SNP typing of free fetal DNA in maternal plasma.