Quantification of Circulating DNA: In the Preparation Lies the Rub

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Abstract
Although cell-free nucleic acids were first described in the 1940s (1), it was not until tumor-specific DNA sequences were detected in the plasma of cancer patients (2) that they started to attract the interest of the wider scientific community. Because the placenta shares numerous features with malignant tissues, such as a high rate of cell turnover and the expression of certain protooncogenes, these seminal reports prompted Lo and colleagues to examine whether fetal DNA could be detected in an analogous manner in the plasma of pregnant women. This hypothesis indeed proved to be true, and in 1997 Lo et al. (3) described the presence of fetal DNA circulating in maternal plasma and serum. This observation has been one of the most important for those attempting to develop safe and efficacious methods for prenatal diagnosis of fetal genetic traits. The reason for this interest is that current methods of obtaining fetal material for prenatal diagnosis rely on invasive procedures such as amniocentesis or chorionic villus sampling, both of which are associated with a procedure-related fetal loss rate of almost 1% (4). Although concerted efforts had been made to develop noninvasive, and hence risk-free, alternative prenatal diagnostic methods using fetal cells enriched from the blood of pregnant women, this approach has been hampered by the extreme scarcity of these cells in the maternal circulation (5). By contrast, fetal DNA could be detected in as little as 10 μL of maternal plasma (3). Consequently, this approach was readily seized on by various research groups, who in rapid succession showed that circulatory fetal DNA can be reliably used for the determination of fetal sex, rhesus D status, and inherited Mendelian disorders [reviewed in Ref. (6)].