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.

Oligonucleotides are unique chemical moieties that can serve as a useful assembly tool. Unlike most bioadhesion molecules that bind together with high affinity, the attraction between complementary DNA strands can vary greatly depending on strand characteristics (e.g., length and sequence choice) and solution conditions (e.g., ionic strength and temperature). We have studied DNA-mediated assembly of micron-sized, bidisperse mixtures using primarily optical and confocal microscopy. To increase hybridization efficiency between complementary strands, DNA sequences were designed to have a low self-affinity that minimizes intrastrand loop and hairpin formations. Single strands of biotinylated DNA were tethered to NeutrAvidin-coated 1.10 and 1.87 micron beads. The resulting oligonucleotide density on the large bead surface was quantified using flow cytometry. In binary mixtures, we found we could vary the degree of binding between complementary beads depending on the number of matching pairs and the ionic strength of the solution. We have also observed a variety of colloidal structures such as chains of alternating large and small particles by exploring additional experimental variables such as particle number ratio and volume fraction.

Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.

We present a novel approach to the fabrication of binary colloidal materials where specific biomolecular cross-linking drives the self-assembly of bidisperse colloidal suspensions. In particular, we have employed low-affinity immune system carbohydrate-selectin interactions to mediate the heterotypic assembly of binary colloidal structures. Using small (0.94 µm) and larger (5.5 µm) diameter particles coated with complementary chemistry, we show that a progressive series of structures, such as colloidal micelles (a large particle coated with smaller particles), colloidal clusters, rings, and elongated chains, can be made by decreasing the number fraction, NA/NB, of small (A) to large (B) particles (200 g NA/NB g 2) at low total volume fraction (φΤ ) 10 -4 -10 -3 ). The assembly is due to specific molecular interactions, as control experiments in which the molecules are blocked or eliminated do not lead to the assembly of these structures. The size of the structures can be modulated by time or total volume fraction. Currently, our methods employ high molecular surface densities, such that the structures result from kinetically trapped, diffusionlimited assembly. Ultimately, with the ability to control the strength of the interaction (using different chemistries and molecular surface densities) as well as the lengths of the molecular tethering arms, particle number densities, and physical properties of the colloidal components, this colloidal assembly driven by specific interactions should yield new materials with many potential technological applications including optical filters, sensors, and separation media.