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...