Noninvasive Prenatal Genetic Testing Using Circulating Cell-Free DNA

Expectant MotherScientists have known for some time that fetal DNA can be detected in the maternal bloodstream during pregnancy (1). Up to 10% of circulating cell-free-DNA (ccfDNA) can be attributed to the fetus. Fetal ccfDNA is released from the placenta, mainly through apoptosis, and enters the maternal bloodstream, where it can be easily collected and detected by PCR amplification. This method of collection has a much lower risk of miscarriage compared to more invasive collection methods such as amniocentesis and chorionic villus sampling.

Amplification of fetal ccfDNA enables a number of prenatal genetic testing such as gender determination and detection of fetal aneuploidy and other mutations. Testing of ccfDNA also allows identification of fetuses with a higher risks of hemolytic disease of the newborn (erythroblastosis fetalis) due to expression of the Rh factor in an Rh– negative mother, who can develop antibodies against the Rh factor and mount an immune response against fetal red blood cells. Finally, ccfDNA allows prenatal paternity determination (2). However, these tests have limitations.

Several factors can complicate the process, including low yields of ccfDNA from blood, relatively high proportions of maternal DNA and short fragment length for fetal DNA—most fetal DNA fragments are <150bp and rarely longer than 250bp (3). Fortunately, researchers have devised ways to overcome or minimize these limitations. For example, starting with a higher volume of maternal plasma can compensate for low ccfDNA yields, as can increasing the number of PCR cycles during amplification. Unfortunately, with increasing cycle numbers comes a higher chance of contamination and artifacts such as nonspecific amplification—not an ideal solution.

A better approach is often to minimize interference by the maternal DNA. For example, the yield of maternal DNA can be reduced during DNA extraction by introducing a size separation step so that the smaller fetal DNA fragments are preferentially isolated. Also, assays often can be designed to focus on paternally inherited genetic loci. A classic example is prenatal paternity testing of a male fetus, which involves amplification of short tandem repeat loci on the Y-chromosome (Y-STRs). These Y-STR loci are male-specific, and thus maternal DNA is not amplified and does not interfere with analysis. Furthermore, PCR primers can be designed to generate smaller Y-STR amplicons for more efficient amplification of fragmented fetal DNA with fewer PCR cycles. In a recent Forensic Science International Genetics paper (4), scientists performing paternity tests using male fetal ccfDNA at 12 weeks gestation had amplification success rates of less than 55% for Y-STR loci with amplicons of >250bp, but their success rate skyrocketed to 100% for mini Y-STR loci (i.e., those with amplicons smaller than 250bp). Using the resulting Y-STR data, they were able to predict fetal gender with 100% accuracy and calculate a probability of paternity of >95.7% in all 20 cases studied.

Even with these improvements and workarounds, prenatal genetic testing still has limitations. Maternal DNA cannot be eliminated entirely during DNA extraction, and assays cannot always be designed so that amplification of only the paternal alleles is informative. Prenatal paternity testing is not possible with women bearing female fetuses and can only narrow paternity to a male lineage, not an individual, because all males of a single lineage inherit the same Y chromosome.

Despite these limitations, the use of circulating cell-free DNA for prenatal genetic testing is quickly replacing more invasive testing approaches, benefitting both mother and fetus due to the reduced risks.


  1. Lo, Y.M. et al. (1997) Presence of fetal DNA in maternal plasma and serum. Lancet 350, 485–7.
  2. Wagner, J. et al. (2009) Non-invasive prenatal paternity testing from maternal blood. J. Legal Med. 123, 75-9.
  3. Fan, H.C. et al. (2010) Analysis of the size distributions of fetal and maternal cell-free DNA by paired-end sequencing. Chem. 56, 1279–86.
  4. Barcelos Barra, G. et al. (2015) Fetal male lineage determination by analysis of Y-chromosome STR haplotype in maternal plasma. Forensic Sci. Int. Genet. 15, 105–110.
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Terri Sundquist

Terri has worked as a Scientific Communications Specialist at Promega Corporation for more than 13 years, and prior to that, spent more than 5 years solving problems and answering questions as a Promega Technical Services Scientist. She graduated with B.S. degrees in Chemistry and Biology at the University of Wisconsin—River Falls, then earned her M.S. in Molecular Biology from the Mayo Graduate School in Rochester Minnesota.

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