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...

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.

A fluorescence-based integrated optics microfluidic device is presented, capable of detecting single DNA molecules in a high throughput and reproducible manner. The device integrates microfluidics for DNA stretching with two optical elements for single molecule detection (SMD): a plano-aspheric refractive lens for fluorescence excitation (illuminator) and a solid parabolic reflective mirror for fluorescence collection (collector). Although miniaturized in size, both optical components were produced and assembled onto the microfluidic device by readily manufacturable fabrication techniques. The optical resolution of the device is determined by the small and relatively low numerical aperture (NA) illuminator lens (0.10 effective NA, 4.0 mm diameter) that delivers excitation light to a diffraction limited 2.0 microm diameter spot at full width half maximum within the microfluidic channel. The collector (0.82 annular NA, 15 mm diameter) reflects the fluorescence over a large collection angle, representing 71% of a hemisphere, toward a single photon counting module in an infinity-corrected scheme. As a proof-of-principle experiment for this simple integrated device, individual intercalated lambda-phage DNA molecules (48.5 kb) were stretched in a mixed elongational-shear microflow, detected, and sized with a fluorescence signal to noise ratio of 9.9 +/-1.0. We have demonstrated that SMD does not require traditional high numerical aperture objective lenses and sub-micron positioning systems conventionally used in many applications. Rather, standard manufacturing processes can be combined in a novel way that promises greater accessibility and affordability for microfluidic-based single molecule applications.