Sequences of PCR primers and probes for real time PCR assays.

<p>Sequences of PCR primers and probes for real time PCR assays.</p

Box plots showing the Ct values for <i>RASSF1A</i>, <i>SRY</i> and <i>DYS14</i> real time PCR assays for male and female pregnancies.

<p>The mean Ct values were 33.9 (<i>RASSF1A</i>, male fetuses), 34.2 (<i>RASSF1A</i>, female fetuses), 33.0 (<i>SRY</i>, male fetuses), 0 (<i>SRY</i>, female fetuses), 32.5 (<i>DYS14</i>, male fetuses) and 39.2 (<i>DYS14</i>, female fetuses).</p

Schematic representation of the possible results obtained using hypermethylated <i>RASSF1A</i> as a universal fetal marker when determining fetal sex by NIPD for a male fetus, female fetus, a case where no cell free fetal DNA is present and a case where no total cell free DNA is present.

<p>Ticks represent the presence of amplified products from either the <i>SRY</i> assay or the <i>RASSF1A</i> assay. Crosses represent no amplification of <i>SRY</i> or <i>RASSF1A</i> amplicons.</p

High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics