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Chungwen Wei, Devereux N Saller, J W H Sutherland, Detection and Quantification by Homogeneous PCR of Cell-free Fetal DNA in Maternal Plasma, Clinical Chemistry, Volume 47, Issue 2, 1 February 2001, Pages 336–338, https://doi.org/10.1093/clinchem/47.2.336
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Prenatal diagnosis of genetic disorders has traditionally relied on invasive procedures such as amniocentesis and chorionic villus sampling (CVS). As these procedures carry a small but significant risk of pregnancy loss, a convenient noninvasive prenatal diagnostic has long been sought. Although fetal cells circulating in the maternal blood have been used for prenatal screening, because of their rarity they require ∼104- to 105-fold enrichment to be detectable (1). Recently, extracellular fetal DNA has been detected in maternal circulation, suggesting that maternal blood might be a useful source of material for noninvasive prenatal diagnosis (2)(3).
To quantify fetal DNA in maternal circulation, we established a quantitative, homogeneous TaqMan PCR assay based on fluorogenic probes (4). This technique involves continuous monitoring of the progress of amplification and permits target quantification. Such analytical characteristics as precision, sensitivity, and dynamic range were analyzed using human genomic DNA as template. The reproducibility of DNA extraction and PCR amplification was also determined using as target cell-free DNA extracted from pooled plasma. This assay was then tested by determining the genders of adult donors by measuring cell-free DNA in their plasma. Finally, using the male-specific SRY gene as a marker for male fetuses, we successfully determined the gender of all fetuses tested.
