Study Of Non-invasive Prenatal Diagnosis Using Cell-free Fetal DNA In Maternal Plasma

Objectives:To establish the technology for extracting cell-free fetal DNA in maternal plasma, comparing detection rate of fetal component marker SRY gene and RASSF1A gene. Utilize the cell-free fetal DNA in maternal plasma associating respectively with traditional nested-PCR method and co-amplification at lower denaturation temperature PCR (COLD-PCR) method to carry on reserch of non-invasive prenatal genetic diagnosis for paternally inherited point mutation of congenita adrenal cortical hyperplasia (CAH)Methods:Extracted plasma DNA from 30 cases of pregnant women' peripheral blood and applied Real-time PCR method in detection of SRY gene and RASSFIA gene for cell-free fetal DNA in it. Extracted plasma DNA from a pregnant woman who had given birth to a boy suffering from CAH and used traditional nested-PCR method and COLD-PCR method respectively to amplify CYP21 gene sequence of cell-free fetal DNA in maternal plasma. Denaturalization high performance liquid chromatography (dHPLC) and restriction analysis were applied in detecting paternally inherited point mutation Q318X of CAH.Results:Of 30 cases of cell-free fetal DNA in maternal plasma SRY gene was detected in 16 cases and detection rate was 88.9%, while RASSF1A gene was detected in 26 cases and detection rate was 86.7%. After amplifying CYP21 gene sequence, paternally inherited point mutation Q318X could be detected from DHPLC and restriction analysis no matter using traditional nested-PCR method or COLD-PCR method. This result was consistent with amniotic fluid prenatal diagnosis. Moreover, mutation detection effect of COLP-PCR method is much better than traditional nested-PCR method after amplification.Conclusions:Establish effective and steady technology for separating and extracting cell-free fetal DNA in maternal plasma. Use cell-free fetal DNA to take non-invasive prenatal diagnosis for a fetus what was on risk of having CAH. It provides a new thinking way of non-invasive prenatal genetic diagnosis for single-gene disorders.