Selected Publications

(Click on citations with a (+) to view the abstract)

1. Ananiev, G. E., Goldstein, S., Runnheim, R., Forrest, D. K., Zhou, S., Potamousis, K., Churas, C. P., Bergendahl, V., Thomson, J. A. and Schwartz, D. C. (2008) “Optical mapping discerns genome wide DNA methylation profiles.” BMC Molecular Biology, accepted.
2. Yu, H and Schwartz, D.C. (2008) “Imaging and analysis of transcription on large, surface-mounted single template DNA molecules.” Analytical Biochemistry, In Press.
3. Kidd, K. M., G. M. Cooper, et al. (2008). "Fine-scale mapping and sequencing of structural variation from eight human genomes." Nature 453(7191): 56-64.
4. Izmitli, A., D. C. Schwartz, et al. (2008). "The effect of hydrodynamic Interactions on the dynamics of DNA translocation through pores." J. of Chem. Phys. 128(8): 085102.
5. Herschleb, J., G. Ananiev, et al. (2007). "Pulsed-Field Gel Electrophoresis." Nature Protocols 2(3): 677-684.
6. Jo, K., D. M. Dhingra, et al. (2007). "A single molecule barcoding system using nanoslits for DNA analysis." Proc Natl Acad Sci USA 104: 2673-2678.
7. Knotts, T. A., N. Rathore, et al. (2007). "A coarse grain model for DNA." J Chem Phys 126: 084901 1-12.
8. Li., H., A. Valouev, et al. (2007). "A quantile method for sizing optical maps." J of Comput Biol. 14: 1-12.
9. Wu, T. and D. C. Schwartz (2007). "Transchip: single-molecule detection of transcriptional elongation complexes " Anal Biochem 361: 31-46.
10. Zhou, S., M. C. Bechner, M. Place, C. P. Churas, L. Pape, S. A. Leong, R. Runnheim, D. K. Forrest, S. Goldstein, M. Livny and D. C. Schwartz (2007). "Validation of rice genome sequence by optical mapping." BMC Genomics 8: 278.
11. Zhou, S., J. Herschleb, et al. (2007). Optical Mapping: A single molecule system for genome analysis. New Methods for DNA Sequencing K. R. Mitchelson, Elsevier Scientific Publishers: 265-300.
12. (+) Valouev, A., L. Li, et al. (2006). "Alignment of optical maps." J Comput Biol 13(2): 442-62.
    We introduce a new scoring method for calculation of alignments of optical maps. Missing cuts, false cuts, and sizing errors present in optical maps are addressed by our alignment score through calculation of corresponding likelihoods. The size error model is derived through the application of Central Limit Theorem and validated by residual plots collected from real data. Missing cuts and false cuts are modeled as Bernoulli and Poisson events, respectively, as suggested by previous studies. Likelihoods are used to derive an alignment score through calculation of likelihood ratios for a certain hypothesis test. This allows us to achieve maximal descriminative power for the alignment score. Our scoring method is naturally embedded within a well known DP framework for finding optimal alignments.
13. (+) Valouev, A., D. C. Schwartz, et al. (2006). "An algorithm for assembly of ordered restriction maps from single DNA molecules." Proc Natl Acad Sci U S A 103(43): 15770-5.
    The restriction mapping of a massive number of individual DNA molecules by optical mapping enables assembly of physical maps spanning mammalian and plant genomes; however, not through computational means permitting completely de novo assembly. Existing algorithms are not practical for genomes larger than lower eukaryotes due to their high time and space complexity. In many ways, sequence assembly parallels map assembly, so that the overlap-layout-consensus strategy, recently shown effective in assembling very large genomes in feasible time, sheds new light on solving map construction issues associated with single molecule substrates. Accordingly, we report an adaptation of this approach as the formal basis for de novo optical map assembly and demonstrate its computational feasibility for assembly of very large genomes. As such, we discuss assembly results for a series of genomes: human, plant, lower eukaryote and bacterial. Unlike sequence assembly, the optical map assembly problem is actually more complex because restriction maps from single molecules are constructed, manifesting errors stemming from: missing cuts, false cuts, and high variance of estimated fragment sizes; chimeric maps resulting from artifactually merged molecules; and true overlap scores that are "in the noise" or "slightly above the noise." We address these problems, fundamental to many single molecule measurements, by an effective error correction method using global overlap information to eliminate spurious overlaps and chimeric maps that are otherwise difficult to identify.
14. (+) Valouev, A., Y. Zhang, et al. (2006). "Refinement of optical map assemblies." Bioinformatics 22(10): 1217-24.
    MOTIVATION: Genomic mutations and variations provide insightful information about the functionality of sequence elements and their association with human diseases. Traditionally, variations are identified through analysis of short DNA sequences, usually shorter than 1000 bp per fragment. Optical maps provide both faster and more cost-efficient means for detecting such differences, because a single map can span over 1 million bp. Optical maps are assembled to cover the whole genome, and the accuracy of assembly is critical. RESULTS: We present a computationally efficient model-based method for improving quality of such assemblies. Our method provides very high accuracy even with moderate coverage (<20 x). We utilize a hidden Markov model to represent the consensus map and use the expectation-Maximization algorithm to drive the refinement process. We also provide quality scores to assess the quality of the finished map. AVAILABILITY: Code is available from www.cmb.usc.edu/people/valouev/
15. (+) Zody, M. C., M. Garber, et al. (2006). "DNA sequence of human chromosome 17 and analysis of rearrangement in the human lineage." Nature 440(7087): 1045-9.
    Chromosome 17 is unusual among the human chromosomes in many respects. It is the largest human autosome with orthology to only a single mouse chromosome, mapping entirely to the distal half of mouse chromosome 11. Chromosome 17 is rich in protein-coding genes, having the second highest gene density in the genome. It is also enriched in segmental duplications, ranking third in density among the autosomes. Here we report a finished sequence for human chromosome 17, as well as a structural comparison with the finished sequence for mouse chromosome 11, the first finished mouse chromosome. Comparison of the orthologous regions reveals striking differences. In contrast to the typical pattern seen in mammalian evolution, the human sequence has undergone extensive intrachromosomal rearrangement, whereas the mouse sequence has been remarkably stable. Moreover, although the human sequence has a high density of segmental duplication, the mouse sequence has a very low density. Notably, these segmental duplications correspond closely to the sites of structural rearrangement, demonstrating a link between duplication and rearrangement. Examination of the main classes of duplicated segments provides insight into the dynamics underlying expansion of chromosome-specific, low-copy repeats in the human genome.
16. Chen, Y. L., M. D. Graham, et al. (2005). "DNA molecules in microfluidic oscillatory flow." Macromolecules 38: 6680-6687.
17. (+) Ivens, A. C., C. S. Peacock, et al. (2005). "The genome of the kinetoplastid parasite, Leishmania major." Science 309(5733): 436-42.
    Leishmania species cause a spectrum of human diseases in tropical and subtropical regions of the world. We have sequenced the 36 chromosomes of the 32.8-megabase haploid genome of Leishmania major (Friedlin strain) and predict 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes, of which 36% can be ascribed a putative function. These include genes involved in host-pathogen interactions, such as proteolytic enzymes, and extensive machinery for synthesis of complex surface glycoconjugates. The organization of protein-coding genes into long, strand-specific, polycistronic clusters and lack of general transcription factors in the L. major, Trypanosoma brucei, and Trypanosoma cruzi (Tritryp) genomes suggest that the mechanisms regulating RNA polymerase II-directed transcription are distinct from those operating in other eukaryotes, although the trypanosomatids appear capable of chromatin remodeling. Abundant RNA-binding proteins are encoded in the Tritryp genomes, consistent with active posttranscriptional regulation of gene expression.
18. (+) Ramanathan, A., L. Pape, et al. (2005). "High-density polymerase-mediated incorporation of fluorochrome-labeled nucleotides." Anal Biochem 337(1): 1-11.
    DNA-polymerase-mediated incorporation of different fluorochrome-labeled nucleotides (FdNTPs) was investigated with the goals of optimizing the high-density labeling of probes and exploring DNA sequencing strategies that rely on the controlled, sequential addition of such compounds. By systematically evaluating variables--including polymerase type, buffer conditions, and fluorochrome chemistries--a rational strategy for the sequential addition of labeled nucleotides to a DNA template was demonstrated. A simple structural model of the polymerase-DNA template complex that considered the fluorochrome moiety of the FdNTPs and the linker length also guided this strategy. Complementary results that portend the use of simple photobleaching to enable the reliable quantitation of consecutive additions are presented.
19. Reslewic, S., J. Herschleb, et al. (2005). "Looking at whole genome one molecule at a time." American Biotechnology Laboratory 23: 26-29.
20. (+) Reslewic, S., S. Zhou, et al. (2005). "Whole-genome shotgun optical mapping of Rhodospirillum rubrum." Appl Environ Microbiol 71(9): 5511-22.
    Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a "molecular cytogenetics" approach to solving problems in genomic analysis.
21. (+) Armbrust, E. V., J. A. Berges, et al. (2004). "The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism." Science 306(5693): 79-86.
    Diatoms are unicellular algae with plastids acquired by secondary endosymbiosis. They are responsible for approximately 20% of global carbon fixation. We report the 34 million-base pair draft nuclear genome of the marine diatom Thalassiosira pseudonana and its 129 thousand-base pair plastid and 44 thousand-base pair mitochondrial genomes. Sequence and optical restriction mapping revealed 24 diploid nuclear chromosomes. We identified novel genes for silicic acid transport and formation of silica-based cell walls, high-affinity iron uptake, biosynthetic enzymes for several types of polyunsaturated fatty acids, use of a range of nitrogenous compounds, and a complete urea cycle, all attributes that allow diatoms to prosper in aquatic environments.
22. (+) Dimalanta, E. T., A. Lim, et al. (2004). "A microfluidic system for large DNA molecule arrays." Anal Chem 76(18): 5293-301.
    Single molecule approaches offer the promise of large, exquisitely miniature ensembles for the generation of equally large data sets. Although microfluidic devices have previously been designed to manipulate single DNA molecules, many of the functionalities they embody are not applicable to very large DNA molecules, normally extracted from cells. Importantly, such microfluidic devices must work within an integrated system to enable high-throughput biological or biochemical analysis-a key measure of any device aimed at the chemical/biological interface and required if large data sets are to be created for subsequent analysis. The challenge here was to design an integrated microfluidic device to control the deposition or elongation of large DNA molecules (up to millimeters in length), which would serve as a general platform for biological/biochemical analysis to function within an integrated system that included massively parallel data collection and analysis. The approach we took was to use replica molding to construct silastic devices to consistently deposit oriented, elongated DNA molecules onto charged surfaces, creating massive single molecule arrays, which we analyzed for both physical and biochemical insights within an integrated environment that created large data sets. The overall efficacy of this approach was demonstrated by the restriction enzyme mapping and identification of single human genomic DNA molecules.
23. Jendrejack, R. M., D. C. Schwartz, et al. (2004). "Shear-induced migration in flowing polymer solutions: Simulation of long-chain deoxyribose nucleic acid in microchannels." J Chem Phys 120: 2513-2529.
24. (+) Ramanathan, A., E. J. Huff, et al. (2004). "An integrative approach for the optical sequencing of single DNA molecules." Anal Biochem 330(2): 227-41.
    A new approach for optically sequencing ensembles of single DNA molecules using DNA polymerase to mediate the consecutive incorporation of fluorochrome-labeled nucleotides into an array of large single DNA molecules is presented. The approach utilizes cycles of labeled fluorochrome addition, detection to count incorporations, and bleaching to reset the counter. These additions are imaged and analyzed to estimate the number of labeled additions and to correlate them on a per-locus basis along DNA backbones. Initial studies used precisely labeled polymerase chain reaction products to aid the development and validation of simple models of fluorochrome point spread functions within the imaging system. In complementary studies, nucleotides labeled with the fluorochrome R110 were incorporated into surface-elongated lambda DNA, and fluorescent signals corresponding to the addition of R110-dUTP were counted and assigned precise loci along DNA backbones. The labeled DNAs were then subjected to photobleaching and to a second cycle of addition of R110-labeled nucleotides-a second round of additions was correlated with the first to establish strings of addition histories among the ensemble of largely double-stranded templates. These results confirm the basic operational validity of this approach and point the way to the development of a practical system for optical sequencing.
25. Schwartz, D. C. (2004). The New Biology. The Markey Scholars Conference Proceedings, (Ed., G.R. Reinhart) National Academies Press: pp.73-79.
26. (+) Zhou, S., A. Kile, et al. (2004). "Single-molecule approach to bacterial genomic comparisons via optical mapping." J Bacteriol 186(22): 7773-82.
    Modern comparative genomics has been established, in part, by the sequencing and annotation of a broad range of microbial species. To gain further insights, new sequencing efforts are now dealing with the variety of strains or isolates that gives a species definition and range; however, this number vastly outstrips our ability to sequence them. Given the availability of a large number of microbial species, new whole genome approaches must be developed to fully leverage this information at the level of strain diversity that maximize discovery. Here, we describe how optical mapping, a single-molecule system, was used to identify and annotate chromosomal alterations between bacterial strains represented by several species. Since whole-genome optical maps are ordered restriction maps, sequenced strains of Shigella flexneri serotype 2a (2457T and 301), Yersinia pestis (CO 92 and KIM), and Escherichia coli were aligned as maps to identify regions of homology and to further characterize them as possible insertions, deletions, inversions, or translocations. Importantly, an unsequenced Shigella flexneri strain (serotype Y strain AMC[328Y]) was optically mapped and aligned with two sequenced ones to reveal one novel locus implicated in serotype conversion and several other loci containing insertion sequence elements or phage-related gene insertions. Our results suggest that genomic rearrangements and chromosomal breakpoints are readily identified and annotated against a prototypic sequenced strain by using the tools of optical mapping.
27. (+) Zhou, S., A. Kile, et al. (2004). "Shotgun optical mapping of the entire Leishmania major Friedlin genome." Mol Biochem Parasitol 138(1): 97-106.
    Leishmania is a group of protozoan parasites which causes a broad spectrum of diseases resulting in widespread human suffering and death, as well as economic loss from the infection of some domestic animals and wildlife. To further understand the fundamental genomic architecture of this parasite, and to accelerate the on-going sequencing project, a whole-genome XbaI restriction map was constructed using the optical mapping system. This map supplemented traditional physical maps that were generated by fingerprinting and hybridization of cosmid and P1 clone libraries. Thirty-six optical map contigs were constructed for the corresponding known 36 chromosomes of the Leishmania major Friedlin genome. The chromosome sizes ranged from 326.9 to 2821.3 kb, with a total genome size of 34.7 Mb; the average XbaI restriction fragment was 25.3 kb, and ranged from 15.7 to 77.8 kb on a per chromosomes basis. Comparison between the optical maps and the in silico maps of sequence drawn from completed, nearly finished, or large sequence contigs showed that optical maps served several useful functions within the path to create finished sequence by: guiding aspects of the sequence assembly, identifying misassemblies, detection of cosmid or PAC clones misplacements to chromosomes, and validation of sequence stemming from varying degrees of finishing. Our results also showed the potential use of optical maps as a means to detect and characterize map segmental duplication within genomes.
28. Zhou, S. and D. Schwartz (2004). "The optical mapping of microbial genomes." ASM News 70: 323-330.
29. (+) El-Sayed, N. M., E. Ghedin, et al. (2003). "The sequence and analysis of Trypanosoma brucei chromosome II." Nucleic Acids Res 31(16): 4856-63.
    We report here the sequence of chromosome II from Trypanosoma brucei, the causative agent of African sleeping sickness. The 1.2-Mb pairs encode about 470 predicted genes organised in 17 directional clusters on either strand, the largest cluster of which has 92 genes lined up over a 284-kb region. An analysis of the GC skew reveals strand compositional asymmetries that coincide with the distribution of protein-coding genes, suggesting these asymmetries may be the result of transcription-coupled repair on coding versus non-coding strand. A 5-cM genetic map of the chromosome reveals recombinational 'hot' and 'cold' regions, the latter of which is predicted to include the putative centromere. One end of the chromosome consists of a 250-kb region almost exclusively composed of RHS (pseudo)genes that belong to a newly characterised multigene family containing a hot spot of insertion for retroelements. Interspersed with the RHS genes are a few copies of truncated RNA polymerase pseudogenes as well as expression site associated (pseudo)genes (ESAGs) 3 and 4, and 76 bp repeats. These features are reminiscent of a vestigial variant surface glycoprotein (VSG) gene expression site. The other end of the chromosome contains a 30-kb array of VSG genes, the majority of which are pseudogenes, suggesting that this region may be a site for modular de novo construction of VSG gene diversity during transposition/gene conversion events.
30. Jendrejack, R. M., E. T. Dimalanta, et al. (2003). "DNA dynamics in a microchannel." Phys Rev Lett 91: 038102(1)-038102(4).
31. Jendrejack, R. M., D. C. Schwartz, et al. (2003). "Effect of confinement on DNA in microfluidic devices." J Chem Phys 119: 1165-1173.
32. (+) Zhou, S., E. Kvikstad, et al. (2003). "Whole-genome shotgun optical mapping of Rhodobacter sphaeroides strain 2.4.1 and its use for whole-genome shotgun sequence assembly." Genome Res 13(9): 2142-51.
    Rhodobacter sphaeroides 2.4.1 is a facultative photoheterotrophic bacterium with tremendous metabolic diversity, which has significantly contributed to our understanding of the molecular genetics of photosynthesis, photoheterotrophy, nitrogen fixation, hydrogen metabolism, carbon dioxide fixation, taxis, and tetrapyrrole biosynthesis. To further understand this remarkable bacterium, and to accelerate an ongoing sequencing project, two whole-genome restriction maps (EcoRI and HindIII) of R. sphaeroides strain 2.4.1 were constructed using shotgun optical mapping. The approach directly mapped genomic DNA by the random mapping of single molecules. The two maps were used to facilitate sequence assembly by providing an optical scaffold for high-resolution alignment and verification of sequence contigs. Our results show that such maps facilitated the closure of sequence gaps by the early detection of nascent sequence contigs during the course of the whole-genome shotgun sequencing process.
33. (+) Zhou, S., W. Deng, et al. (2002). "A whole-genome shotgun optical map of Yersinia pestis strain KIM." Appl Environ Microbiol 68(12): 6321-31.
    Yersinia pestis is the causative agent of the bubonic, septicemic, and pneumonic plagues (also known as black death) and has been responsible for recurrent devastating pandemics throughout history. To further understand this virulent bacterium and to accelerate an ongoing sequencing project, two whole-genome restriction maps (XhoI and PvuII) of Y. pestis strain KIM were constructed using shotgun optical mapping. This approach constructs ordered restriction maps from randomly sheared individual DNA molecules directly extracted from cells. The two maps served different purposes; the XhoI map facilitated sequence assembly by providing a scaffold for high-resolution alignment, while the PvuII map verified genome sequence assembly. Our results show that such maps facilitated the closure of sequence gaps and, most importantly, provided a purely independent means for sequence validation. Given the recent advancements to the optical mapping system, increased resolution and throughput are enabling such maps to guide sequence assembly at a very early stage of a microbial sequencing project.
34. (+) Lim, A., E. T. Dimalanta, et al. (2001). "Shotgun optical maps of the whole Escherichia coli O157:H7 genome." Genome Res 11(9): 1584-93.
    We have constructed NheI and XhoI optical maps of Escherichia coli O157:H7 solely from genomic DNA molecules to provide a uniquely valuable scaffold for contig closure and sequence validation. E. coli O157:H7 is a common pathogen found in contaminated food and water. Our approach obviated the need for the analysis of clones, PCR products, and hybridizations, because maps were constructed from ensembles of single DNA molecules. Shotgun sequencing of bacterial genomes remains labor-intensive, despite advances in sequencing technology. This is partly due to manual intervention required during the last stages of finishing. The applicability of optical mapping to this problem was enhanced by advances in machine vision techniques that improved mapping throughput and created a path to full automation of mapping. Comparisons were made between maps and sequence data that characterized sequence gaps and guided nascent assemblies.
35. Aston, C., C. Hiort, et al. (1999). "Optical mapping: an approach for fine mapping." Methods Enzymol 303: 55-73.
36. (+) Aston, C., B. Mishra, et al. (1999). "Optical mapping and its potential for large-scale sequencing projects." Trends Biotechnol 17(7): 297-302.
    Physical mapping has been rediscovered as an important component of large-scale sequencing projects. Restriction maps provide landmark sequences at defined intervals, and high-resolution restriction maps can be assembled from ensembles of single molecules by optical means. Such optical maps can be constructed from both large-insert clones and genomic DNA, and are used as a scaffold for accurately aligning sequence contigs generated by shotgun sequencing.
37. (+) Jing, J., Z. Lai, et al. (1999). "Optical mapping of Plasmodium falciparum chromosome 2." Genome Res 9(2): 175-81.
    Detailed restriction maps of microbial genomes are a valuable resource in genome sequencing studies but are toilsome to construct by contig construction of maps derived from cloned DNA. Analysis of genomic DNA enables large stretches of the genome to be mapped and circumvents library construction and associated cloning artifacts. We used pulsed-field gel electrophoresis purified Plasmodium falciparum chromosome 2 DNA as the starting material for optical mapping, a system for making ordered restriction maps from ensembles of individual DNA molecules. DNA molecules were bound to derivatized glass surfaces, cleaved with NheI or BamHI, and imaged by digital fluorescence microscopy. Large pieces of the chromosome containing ordered DNA restriction fragments were mapped. Maps were assembled from 50 molecules producing an average contig depth of 15 molecules and high-resolution restriction maps covering the entire chromosome. Chromosome 2 was found to be 976 kb by optical mapping with NheI, and 946 kb with BamHI, which compares closely to the published size of 947 kb from large-scale sequencing. The maps were used to further verify assemblies from the plasmid library used for sequencing. Maps generated in silico from the sequence data were compared to the optical mapping data, and good correspondence was found. Such high-resolution restriction maps may become an indispensable resource for large-scale genome sequencing projects.
38. (+) Lai, Z., J. Jing, et al. (1999). "A shotgun optical map of the entire Plasmodium falciparum genome." Nat Genet 23(3): 309-13.
    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).
39. (+) Lin, J., R. Qi, et al. (1999). "Whole-genome shotgun optical mapping of Deinococcus radiodurans." Science 285(5433): 1558-62.
    A whole-genome restriction map of Deinococcus radiodurans, a radiation-resistant bacterium able to survive up to 15,000 grays of ionizing radiation, was constructed without using DNA libraries, the polymerase chain reaction, or electrophoresis. Very large, randomly sheared, genomic DNA fragments were used to construct maps from individual DNA molecules that were assembled into two circular overlapping maps (2.6 and 0.415 megabases), without gaps. A third smaller chromosome (176 kilobases) was identified and characterized. Aberrant nonlinear DNA structures that may define chromosome structure and organization, as well as intermediates in DNA repair, were directly visualized by optical mapping techniques after gamma irradiation.
40. (+) Skiadas, J., C. Aston, et al. (1999). "Optical PCR: genomic analysis by long-range PCR and optical mapping." Mamm Genome 10(10): 1005-9.
    Optical mapping is an approach for the rapid, automated, non-electrophoretic construction of ordered restriction maps of DNA from ensembles of single molecules. Previously, we used optical mapping to make high-resolution maps of large insert clones such as bacterial artificial chromosomes (BAC) and large genomic DNA molecules. Here, we describe a combination of optical mapping and long-range polymerase chain reaction (PCR), in a process we term optical PCR, which enables automated construction of ordered restriction maps of long-range PCR products spanning human genomic loci. Specifically, we amplified three long PCR products, each averaging 14.6 kb in length, which span the 37-kb human tissue plasminogen activator (TPA) gene. PCR products were surface mounted in gridded arrays, and samples were mapped in parallel with either ScaI, XmnI, HpaI, ClaI, or BglII. A contig of overlapping high-resolution maps was generated, which agreed closely with maps predicted from sequence data. The data demonstrate an approach to construct physical maps of genomic loci where very little prior sequence information exists, since the only sequence needed is that required to anchor PCR primers. Large segments of genomic DNA (within the practical limits imposed by long-range PCR) can be mapped quickly and to high resolution without the use of cloning vectors.
41. (+) Cai, W., J. Jing, et al. (1998). "High-resolution restriction maps of bacterial artificial chromosomes constructed by optical mapping." Proc Natl Acad Sci U S A 95(7): 3390-5.
    Large insert clone libraries have been the primary resource used for the physical mapping of the human genome. Research directions in the genome community now are shifting direction from purely mapping to large-scale sequencing, which in turn, require new standards to be met by physical maps and large insert libraries. Bacterial artificial chromosome libraries offer enormous potential as the chosen substrate for both mapping and sequencing studies. Physical mapping, however, has come under some scrutiny as being "redundant" in the age of large-scale automated sequencing. We report the development and applications of nonelectrophoretic, optical approaches for high-resolution mapping of bacterial artificial chromosome that offer the potential to complement and thereby advance large-scale sequencing projects.
42. (+) Jing, J., J. Reed, et al. (1998). "Automated high resolution optical mapping using arrayed, fluid-fixed DNA molecules." Proc Natl Acad Sci U S A 95(14): 8046-51.
    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.
43. Anantharaman, T. S., B. Mishra, et al. (1997). "Genomics via optical mapping II: Ordered restriction maps." J Comput Biol 4: 91-118.
44. (+) Schwartz, D. C. and A. Samad (1997). "Optical mapping approaches to molecular genomics." Curr Opin Biotechnol 8(1): 70-4.
    A variety of physical mapping methods exist for the analysis of nucleic acids or genomes, including hybridization, sequence tagged site mapping, restriction enzyme fingerprinting, radiation hybrid mapping and optical mapping. Single-molecule approaches offer numerous advantages, including very high resolution, small sample size requirements, and parallel sample processing. The convergence of recent advances in new single molecule techniques, surface chemistry and machine vision technology has contributed to novel approaches to genome analysis.
45. (+) Cai, W., H. Aburatani, et al. (1995). "Ordered restriction endonuclease maps of yeast artificial chromosomes created by optical mapping on surfaces." Proc Natl Acad Sci U S A 92(11): 5164-8.
    We have developed a surface mounting technology for the rapid construction of ordered restriction maps from individual DNA molecules. Optical restriction maps constructed from yeast artificial chromosome DNA molecules mounted on specially derivatized glass surfaces are accurate and reproducible, and the technology is amenable to automation. The mounting procedures described here should also be useful for fluorescence in situ hybridization studies. We believe these improvements to optical mapping will further stimulate the development of nonelectrophoretic approaches to genome analysis.
46. (+) Meng, X., K. Benson, et al. (1995). "Optical mapping of lambda bacteriophage clones using restriction endonucleases." Nat Genet 9(4): 432-8.
    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.
47. Samad, A., E. F. Huff, et al. (1995). "Optical mapping: a novel, single-molecule approach to genomic analysis." Genome Res 5(1): 1-4.
48. Samad, A. H., W. W. Cai, et al. (1995). "Mapping the genome one molecule at a time--optical mapping." Nature 378(6556): 516-7.
49. (+) Wang, Y. K., E. J. Huff, et al. (1995). "Optical mapping of site-directed cleavages on single DNA molecules by the RecA-assisted restriction endonuclease technique." Proc Natl Acad Sci U S A 92(1): 165-9.
    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.
50. (+) Schwartz, D. C., X. Li, et al. (1993). "Ordered restriction maps of Saccharomyces cerevisiae chromosomes constructed by optical mapping." Science 262(5130): 110-4.
    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.
51. (+) Schwartz, D. C. and M. Koval (1989). "Conformational dynamics of individual DNA molecules during gel electrophoresis." Nature 338(6215): 520-2.
    Gel electrophoresis is widely used in molecular biology to separate DNA molecules according to their sizes. The physical basis of this size separation is, however, poorly understood. Here we report observations of individual, fluorescently stained DNA molecules as they migrate during various kinds of gel electrophoresis. Their movement, under the influence of either a steady electric field or a pulsed-field, is characterized by cycles of elongation and contraction. Initially relaxed coils of DNA lengthen into 'hook-shaped' configurations which temporarily 'hang-up' on obstacles in the gel matrix before sliding off, contracting and entering another cycle. The effects of a new electrophoresis technique, termed 'pulse-oriented electrophoresis', which allows the effective angle of the electric field, and hence the molecular orientation of DNA, to be varied without electrode rearrangement, are also studied. In this case the DNA adopts a 'staircase' configuration showing that the net orientation in a direction is given by the vector sum of the pulses used.
52. (+) Schwartz, D. C. and C. R. Cantor (1984). "Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis." Cell 37(1): 67-75.
    A new type of gel electrophoresis separates DNA molecules up to 2000 kb with resolutions exceeding the logarithmic molecular weight dependence of conventional electrophoresis. The technique uses 1.5% agarose, 10 to 20 micrograms of DNA per well, and low ionic strength buffers. It employs alternately pulsed, perpendicularly oriented electrical fields, at least one of which is inhomogeneous. The duration of the applied electrical pulses is varied from 1 sec to 90 sec to achieve optimal separations for DNAs with sizes from 30 to 2000 kb. This pulsed field gradient gel electrophoresis fractionates intact S. cerevisiae chromosomal DNA, producing a molecular karyotype that greatly facilitates the assignment of genes to yeast chromosomes. Each yeast chromosome consists of a single piece of DNA; the chromosome sizes are consistent with the genetic linkage map. We also describe a general method for preparing spheroplasts, and cell lysates, without significant chromosomal DNA breakage.