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...

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease that results from an oligodendrocyte infection caused by JC virus. The JC virus early promoter directs cell-specific expression of the viral replication factor large T antigen, and thus transcriptional regulation constitutes a major mechanism of glial tropism in PML. We have previously demonstrated that T antigen controls the JC virus basal promoter in a glial cell-specific manner, since T antigen repressed the JC virus and simian virus 40 (SV40) early promoters in glioma cells but induced strong activation of the JC virus early promoter in nonglial cells. To further analyze these findings, T antigen and nuclear extracts from glial and nonglial cells were used to examine DNase I footprints on the proximal promoter. T-antigen binding to site II was more extensive than expected based on sequence homology with SV40, and nuclear proteins protected several regions of the proximal promoter in a cell-specific manner. Multiple Sp1 binding domains were identified. Site-directed mutagenesis revealed that T-antigen-mediated activation required a TATA box sequence, a pentanucleotide repeat immediately upstream of the TATA box, and an Sp1 binding site downstream of the TATA box. When footprints were obtained with mutant promoters which blocked T-antigen-induced transactivation, no change in T-antigen binding was observed. These results suggest that T antigen activates the JC virus basal promoter in nonglial cells by interaction with the transcription initiation complex.The human polyomavirus JC virus is the etiologic agent of progressive multifocal leukoencephalopathy (PML). JC virus selectively destroys oligodendrocytes, leading to multiple areas of demyelination and attendant loss of brain function (3, 31). Once a rare condition, PML is no longer infrequent, occurring in 5% of individuals with AIDS (4). JC virus infection exists in a persistent state in kidney tissue and peripheral blood lymphocytes throughout the life of healthy individuals. In the setting of immunodeficiency, the virus infects and destroys oligodendrocytes, producing patches of myelin loss in subcortical white matter (22). Thus, the neuropathological features suggest that reactivated JC virus infection is specific for glial cells.The JC virus early promoter directs cell-specific expression of the large T antigen, which is required for viral replication, and thus transcriptional regulation constitutes a major mechanism of glial tropism of PML (15). The MH1 form of the JC virus promoter was directly isolated from the brain of a PML patient. Its sequence diverges somewhat from that of the original Mad-1 promoter (11); however, both MH1 and Mad-1 proximal promoters have sequence identity from the initiating codon for T antigen up to a pentanucleotide site just upstream of the viral TATA sequence. The pentanucleotide repeat sequence in MH1 varies by a single base from the perfect repeats seen in the Mad-1 variant of the promoter. This sequence conservation implies that this regi...