Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(®) assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(®) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(®) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.

Targeted sequencing of specific loci of the human genome is a promising approach for maximizing the efficiency of second-generation sequencing technologies for population-based studies of genetic variation. Here we describe microdroplet PCR, which performs 1.5 million separate amplifications in parallel, as an approach for enriching targeted sequences in the human genome. We initially designed primers to 435 exons of 47 genes that were selected for having a broad spectrum of sequence characteristics. Using this primer set we amplified the same six samples by both microdroplet and traditional singleplex PCR and sequenced the products using the Illumina GAII demonstrating that both methods generate similarly high quality data; 84% of the uniquely mapping reads fell within the targeted sequences, uniform coverage of ~90% of the targeted bases, greater than 99% accuracy in sequence variant calls, and high reproducibility between different samples (r2=0.9). We next scaled the microdroplet PCR to 3976 amplicons totaling 1.49 Mb of sequence, sequenced the resulting sample on both the Illumina GAII and Roche 454 platforms, and obtained data with equally high specificity and sensitivity quality. Our results demonstrate that microdroplet technology is well suited for processing DNA for massively parallel amplification of specific subsets of the human genome for targeted sequencing.

We have developed a rapid molecular mapping technology-Direct Linear Analysis (DLA)-on the basis of the analysis of individual DNA molecules bound with sequence-specific fluorescent tags. The apparatus includes a microfluidic device for stretching DNA molecules in elongational flow that is coupled to a multicolor detection system capable of single-fluorophore sensitivity. Double-stranded DNA molecules were tagged at sequence-specific motif sites with fluorescent bisPNA (Peptide Nucleic Acid) tags. The DNA molecules were then stretched in the microfluidic device and driven in a flow stream past confocal fluorescence detectors. DLA provided the spatial locations of multiple specific sequence motifs along individual DNA molecules, and thousands of individual molecules could be analyzed per minute. We validated this technology using the 48.5 kb phage genome with different 8-base and 7-base sequence motif tags. The distance between the sequence motifs was determined with an accuracy of ±0.8 kb, and these tags could be localized on the DNA with an accuracy of ±2 kb. Thus, DLA is a rapid mapping technology, suitable for analysis of long DNA molecules.[Supplemental material is available online at www.genome.org.]Traditionally, DNA mapping has been an important strategy to study structures and organizations of genomes. Recent advances in DNA sequencing technologies, however, have served to diminish the relative importance of traditional mapping. Nonetheless, growing interest in comparative genomics has created a need for technologies that can rapidly and efficiently characterize a genome, particularly larger genomes. Furthermore, in many cases, single-base resolution is unnecessary, as genomic differences among species (e.g., microorganisms) or among individuals within a given species (e.g., humans) can be discerned using lower-resolution mapping approaches (Olive and Bean 1999). Thus, there is a need for a practical, rapid, and highly efficient DNA mapping technology.Currently, restriction mapping is the most practicable mapping approach that combines high resolution with high density (Brown 1999). Gel electrophoresis-based restriction enzyme mapping using just a single enzyme has been a workhorse for the human genome project and other large-scale efforts to provide a fingerprint identification of BAC clones (Soderlund et al. 2000). Traditional restriction mapping with multiple enzymes has allowed characterization and manipulation of genomic regions of interest (Brown 1999). To study human variation, restriction fragment length polymorphism (RFLP) analysis has allowed investigators to identify SNPs that correlate with disease loci (Shi 2002). Nonetheless, restriction mapping has fundamental drawbacks that limit its utility for comparative genomics. Digestion of the DNA removes information regarding the ordering of the fragments, requiring the use of multiple enzymes to construct the correct map. Furthermore, as RFLP analysis involves a mixture of molecules, haplotype information is inaccessible. For large DNA, pulsed-fi...

Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented.

High-throughput stretching and monitoring of single DNA molecules in continuous elongational flow offers compelling advantages for biotechnology applications such as DNA mapping. However, the polymer dynamics in common microfluidic implementations are typically complicated by shear interactions. These effects were investigated by observation of fluorescently labeled 185 kb bacterial artificial chromosomes in sudden mixed shear and elongational microflows generated in funneled microfluidic channels. The extension of individual free DNA molecules was studied as a function of accumulated fluid strain and strain rate. Under constant or gradually changing strain rate conditions, stretching by the sudden elongational component proceeded as previously described for an ideal elongational flow (T. T. Perkins, D. E. Smith and S. Chu, Science, 1997, 276, 2016): first, increased accumulated fluid strain and increased strain rate produced higher stretching efficiencies, despite the complications of shear interactions; and second, the results were consistent with unstretched molecules predominantly in hairpin conformations. More abrupt strain rate profiles did not deliver a uniform population of highly extended molecules, highlighting the importance of balance between shear and elongational components in the microfluidic environment for DNA stretching applications. DNA sizing with up to 10% resolution was demonstrated. Overall, the device delivered 1000 stretched DNA molecules per minute in a method compatible with diffraction-limited optical sequence motif mapping and without requiring laborious chemical modifications of the DNA or the chip surface. Thus, the method is especially well suited for genetic characterization of DNA mixtures such as in pathogen fingerprinting amidst high levels of background DNA.