Molecular confinement offers new routes for arraying large DNA molecules, enabling single-molecule schemes aimed at the acquisition of sequence information. Such schemes can rapidly advance to become platforms capable of genome analysis if elements of a nascent system can be integrated at an early stage of development. Integrated strategies are needed for surmounting the stringent experimental requirements of nanoscale devices regarding fabrication, sample loading, biochemical labeling, and detection. We demonstrate that disposable devices featuring both micro-and nanoscale features can greatly elongate DNA molecules when buffer conditions are controlled to alter DNA stiffness. Furthermore, we present analytical calculations that describe this elongation. We also developed a complementary enzymatic labeling scheme that tags specific sequences on elongated molecules within described nanoslit devices that are imaged via fluorescence resonance energy transfer. Collectively, these developments enable scaleable molecular confinement approaches for genome analysis.
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.
Background: Rice feeds much of the world, and possesses the simplest genome analyzed to date within the grass family, making it an economically relevant model system for other cereal crops. Although the rice genome is sequenced, validation and gap closing efforts require purely independent means for accurate finishing of sequence build data.
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.There are 11 species in the genus Yersinia, 3 of which are pathogenic for humans (Yersinia pestis, Y. enteroclitica, and Y. pseudotuberculosis). Of the three pathogenic species, Y. pestis, which lives in rodents, transmits this pathogen to humans via fleas, causing bubonic, septicemic, and pneumonic plagues (also known as black death). Y. pestis has been responsible for many recurrent devastating pandemics throughout history, resulting in widespread loss of human life (1,7,22). To understand the molecular mechanisms that define the pathogenicity of this bacterial species, sequencing of two strains (KIM and CO-92 biovar Orientalis) of Y. pestis was funded by the National Institute of Allergy and Infectious Diseases and Beowulf Genomics. The two separate sequencing projects were conducted by F. Blattner's laboratory (University of WisconsinMadison) and Sanger Center (Hinxton, United Kingdom), using the strategy of whole-genome shotgun sequencing (11). Although this sequencing strategy has racked up an impressive number of completed microbial genomes, it can be further optimized in terms of the cost and effort required during the finishing stages. In this regard, physical maps serve to guide sequence assembly, characterize gaps, and validate the finished sequence. Furthermore, ordered restriction maps are particularly useful when attempting to assemble genomic regions containing repeats, since cleavage patterns can accurately discern such sequence elements.The costly appellation of "finished," assumed when dealing with high-quality sequence data, minimally mandates that no genomic region be excluded from the final results (23) and thus requires extensive finishing efforts. The chances that entire regions of a genome might be excluded from "completed" sequence increases in the face of limited budgets and the absence of physical mapping data. Because of these issues, optical maps were used (15) at an early stage of sequence assembly to iden...
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.
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