Detection And Analysis Of Cell-free Fetal Dna In Maternal Plasma

Objects:To establish an approach for non-invasive prenatal diagnosis which independent from fetal sex and paternal control, and also can be used for detection of fetal chromosomal aneuploidy and single gene mutations using cell-free fetal DNA in maternal plasma combined with maternal control.Methods:41 peripheral blood samples were obtained from healthy pregnant women. Cell-free fetal DNA was extracted from maternal plasma. DNA extracted from their oral swabs and white blood cells was taken as meternal control. 17 husbands' oral swabs and 4 new-borns' umbilical cord blood samples were volunteered to offer as verification group. Three highly polymorphic short tandem repeat (STR) loci: D17S1293, D21S11, DXS8377 together with the SRY (sex-detennining region on Y) gene were amplified by PCR, Boster PCR, or touch-down PCR according to different sources of DNA samples. Allelic discrimination analysis of three STR loci was investigated through denaturing polyacrylamide gel electrophoresis and silver-staining, while SRY gene was examined by agarose gel electrophoresis. Samples from the same woman or the same family were compared to detect the paternally inherited fetal alleles. Both SRY gene and X-STR locus were used for the determination of fetal sex.Results: 1. Non-maternal inherited alleles were detected from each of the 41 maternal plasma DNA sample. 17 cases of them were verified by paternal DNA, and 4 were verified by fetal DNA, which demonstrated that the non-maternal inherited alleles were paternally inherited fetal alleles.2. Fetal sex was detected by both SRY gene and X-STR locus. 38 of the 41 cases were identified, with 10 of them were verified by new-borns.Conclusions:1. Analysis of cell-free fetal DNA in maternal plasma at SRYgene and DXS8377 locus could get paternally inherited information in both male and female fetuses and could be used in prenatal diagnosis of sex-linked disorders.2. Analysis of cell-free fetal DNA in maternal plasma at autosomal STR loci could be used in prenatal diagnosis of autosomal disorders.3. Touch-down PCR and Boster PCR assays are robust in detecting low-level fetal DNA concentrations during early pregancy.