Zhongwu Lai

A shotgun optical map of the entire Plasmodium falciparum genome

The unicellular parasite Plasmodium falciparum is the cause of human malaria, resulting in 1.7–2.5 million deaths each year. To develop new means to treat or prevent malaria, the Malaria Genome Consortium was formed to sequence and annotate the entire 24.6-Mb genome. The plan, already underway, is to sequence libraries created from chromosomal DNA separated by pulsed-field gel electrophoresis (PFGE). The AT-rich genome of P. falciparum presents problems in terms of reliable library construction and the relative paucity of dense physical markers or extensive genetic resources. To deal with these problems, we reasoned that a high-resolution, ordered restriction map covering the entire genome could serve as a scaffold for the alignment and verification of sequence contigs developed by members of the consortium. Thus optical mapping was advanced to use simply extracted, unfractionated genomic DNA as its principal substrate. Ordered restriction maps (BamHI and NheI) derived from single molecules were assembled into 14 deep contigs corresponding to the molecular karyotype determined by PFGE (ref. 3).

New approaches to genomic analysis using single molecules

Current moIecuIar bioIogy techniques were deveIoped primarily for characterization of single genes, not entire genomes, and, as such, are not ideally suited to high resolution analysis of complex traits and the moiecular genetics of very large populations. Despite rapid progress in the human genome project effort, there is little doubt that radicaIIy new conceptual approaches are needed before routine whole genome-based analyses can be undertaken by both basic research and clinical laboratories. Physical mapping of genomes, using restriction endonucleases, has played a major role in the identification and characterizing various loci, for example, by aiding clone contig formation and by characterizing genetic lesions. Restriction maps provide precise genomic distances, unlike ordered sequencebased landmarks such as Sequence Tagged Sites (ST%), that are essential for optimizing the efficiency of sequencing efforts, and for determining the spatial relationships of specific loci. When compared to tedious hybridization-based fingerprinting approaches, ordered restriction maps offer relatively unambiguous clone characterization that is useful in contig formation, establishment of minimal tiling paths for sequencing, and preliminary characterization of sequence lesions. In addition, such maps provide a useful scaffold for sequence assembly, often critical in the final sequence finishing stage. Despite the broad applications of restriction maps, the associated techniques for their generation have changed little over the last ten years, primarily because they still utilize electrophoretic analysis. To help overcome these shortcomings, our laboratory developed the first practical non-electrophoretic genomic mapping approach, Optical Mapping, to meet this need. Optical Mapping is a single molecule methodology for the rapid production of ordered restriction