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).

Dynamics of DNA molecules in gel studied by fluorescence microscopy.

The dynamics of individual DNA molecules in a thin gel were studied with fluorescence microscopy. Driven by an electric field, molecules hooked around isolated obstacles and became extended. By analyzing molecular images, we identified the reptation tube and primitive chain. When the field was turned off, the molecules relaxed. The relaxation time tau1 and primitive chain length at equilibrium depend on N, the size of the molecule in base pairs, consistently with reptation theory. Using five yeast chromosomal DNAs ranging in size from 245 kb to 980 kb, we found that: These results constitute a way of sizing individual DNA molecules by imaging rather than by gel electrophoresis.

Automated high resolution optical mapping using arrayed, fluid-fixed DNA molecules.

New mapping approaches construct ordered restriction maps from fluorescence microscope images of individual, endonuclease-digested DNA molecules. In optical mapping, molecules are elongated and fixed onto derivatized glass surfaces, preserving biochemical accessibility and fragment order after enzymatic digestion. Measurements of relative fluorescence intensity and apparent length determine the sizes of restriction fragments, enabling ordered map construction without electrophoretic analysis. The optical mapping system reported here is based on our physical characterization of an effect using fluid flows developed within tiny, evaporating droplets to elongate and fix DNA molecules onto derivatized surfaces. Such evaporation-driven molecular fixation produces well elongated molecules accessible to restriction endonucleases, and notably, DNA polymerase I. We then developed the robotic means to grid DNA spots in well defined arrays that are digested and analyzed in parallel. To effectively harness this effect for high-throughput genome mapping, we developed: (i) machine vision and automatic image acquisition techniques to work with fixed, digested molecules within gridded samples, and (ii) Bayesian inference approaches that are used to analyze machine vision data, automatically producing high-resolution restriction maps from images of individual DNA molecules. The aggregate significance of this work is the development of an integrated system for mapping small insert clones allowing biochemical data obtained from engineered ensembles of individual molecules to be automatically accumulated and analyzed for map construction. These approaches are sufficiently general for varied biochemical analyses of individual molecules using statistically meaningful population sizes.

Optical mapping of lambda bacteriophage clones using restriction endonucleases.

Optical mapping is an emerging single molecule approach for the rapid generation of ordered restriction maps, using fluorescence microscopy. We have improved the size resolution of optical mapping by imaging individual DNA molecules elongated and fixed onto derivatized glass surfaces. Averaged fluorescence intensity and apparent length measurements accurately determined the mass of restriction fragments 800 basepairs long. We have used optical mapping to create ordered restriction maps for lambda clones derived from the mouse pygmy locus.

Optical mapping of site-directed cleavages on single DNA molecules by the RecA-assisted restriction endonuclease technique.

Fluorescence in situ hybridization (FISH) resolution has advanced because newer techniques use increasingly decondensed chromatin. FISH cannot analyze restriction enzyme cutting sites due to limitations of the hybridization and detection technologies. The RecA-assisted restriction endonuclease (RARE) technique cleaves chromosomal DNA at a single EcoRI site within a given gene or selected sequence. We recently described a mapping technique, optical mapping, which uses fluorescence microscopy to produce high-resolution restriction maps rapidly by directly imaging restriction digestion cleavage events occurring on single deproteinized DNA molecules. Ordered maps are then constructed by noting fragment order and size, using several optically based techniques. Since we also wanted to map arbitrary sequences and gene locations, we combined RARE with optical mapping to produce site-specific visible EcoRI restriction cleavage sites on single DNA molecules. Here we describe this combined method, named optical RARE, and its initial application to mapping gene locations on yeast chromosomes.

Ordered restriction maps of Saccharomyces cerevisiae chromosomes constructed by optical mapping.

A light microscope-based technique for rapidly constructing ordered physical maps of chromosomes has been developed. Restriction enzyme digestion of elongated individual DNA molecules (about 0.2 to 1.0 megabases in size) was imaged by fluorescence microscopy after fixation in agarose gel. The size of the resulting individual restriction fragments was determined by relative fluorescence intensity and apparent molecular contour length. Ordered restriction maps were then created from genomic DNA without reliance on cloned or amplified sequences for hybridization or analytical gel electrophoresis. Initial application of optical mapping is described for Saccharomyces cerevisiae chromosomes.

Sizing of large DNA molecules by hook formation in a loose matrix.

We present details on how to implement a newly developed methodology, Optical Contour Maximization (OCM), for accurately sizing large DNA molecules. Agarose gel containing stained DNA is cast between a slide and a coverslip, and molecules moved by an applied electrical field are observed using fluorescence microscopy. The molecules of interest lie in the interface formed between the coverslip and gel. DNA movement in this region is largely confined to lateral motions. When a DNA molecule snags an obstacle, it elongates, forming a metastable hook that can persist for several seconds. We found that the longest observed hook contour length can be determined from rapidly collected images. This maximized length shows a linear correlation with reported size [X.H. Guo, E.J. Huff & D.C. Schwartz, Nature 359, 783 (1992)] Successful measurements require a critical balance between the voltage needed for full elongation, and the unwanted effects of too large a voltage: fewer metastable hooks form, they dissipate faster, molecule breakage becomes a problem, and faster image collection becomes necessary. We measured apparent contour length as a function of applied voltage and determined an optimal voltage for our apparatus, using a singly anchored 114kb molecule. The measurement precision is estimated from the distribution of results. We expect that OCM will find utility in physical mapping and molecular karyotyping of lower eucaryotes of medical importance.