Single molecule systems: Advancements and applications to microbial and human genome analysis.

摘要

Optical Mapping is an advanced system for the rapid construction of ordered restriction maps from individual surface-mounted DNA molecules following endonuclease digestion. Whole genome Optical Maps can provide scaffolds to facilitate sequence assembly, gap closure and identify gross chromosomal aberrations. A high-resolution map of E. coli O157 was generated to aid sequence assembly and to mediate gap closure operations. More recently, advancements in the Optical Mapping System have enabled the rapid analysis of complex genomes, and Fluorescence Activated Cell Sorter (FACS) instrument was used isolate human Y chromosomal DNA samples for high-resolution mapping. This new genome analysis approach, Directed Optical Mapping, combines the unique separations offered by the FACS to greatly enrich any sortable chromosomal material. Although FACS preparative yields are notoriously low, and rife with fragmented DNA molecules, these issues were mitigated by the development of novel DNA handling approaches, which produced sufficient yields of large DNA molecules, that were matched by the capabilities of a single molecule platform like Optical Mapping. These efforts made possible a map covering the entire human Y chromosome, which was analyzed to identify a unique set of mutations. This work, plus the single molecule analysis of the E. coli O157 genome, then served as a springboard for the rapid mapping of the entire human genome. This map was analyzed to reveal heterochromatic genomic regions, discern polymorphisms, and detect chromosomal aberrations. In summary, the work presented here points the way to new approaches for the detailed genomic analysis of human populations at the whole genome level.