Microdissection and microcloning of human chromosome 21

Abstract

Detailed physical mapping of specific genomic regions require many DNA probes isolated from the region of interest. A more direct approach is to physically dissect the region and to clone the dissected DNA sequences in small volume using a microcloning procedure. Metaphase chromosome preparations can be used for microdissection. The dissected chromosome fragments containing picograms of DNA are treated with proteinase K/SDS and EcoR1, and ligated to phage vector arms. All these microcloning steps are performed under the microscope in nanoliter volumes. After ligation, the extract is in vitro packaged and plated on selective bacterial host. The recombinant microclones are isolated and characterized for use as probes for physical mapping and molecular studies of the dissected region. These microtechniques have been developed and applied to human chromosome 21. This approach appears to be particularly useful in saturation cloning and mapping of refined chromosomal regions to facilitate search for disease genes mapped to a particular region.