The development of novel techniques permitting one to cross large distances along chromosomes is a crucial part of current efforts to develop complete physical maps of the human genome. Such techniques are also essential to the related goal of cloning genes for human diseases by knowledge of their map position, known as """"""""reverse genetics."""""""" We have recently developed a chromosome jumping technique which depends on the circularization of large genomic fragments, followed by selective cloning of the junctions of these circles. The technique has been successfully carried out for jumps of 100 and 200 kb. In this extension of that work, we propose to construct general jumping libraries of 400 kb and larger jumpsize from both man and mouse. Specific jumping and linking libraries which connect adjacent rare restriction sites (NotI, BssHII, MluI) will also be constructed. We will specifically address some of the technical constraints on the construction of libraries by: 1) using a lac operator sequence to mark the junction fragments, which will allow purification by binding to lac repressor prior to cloning as well as additional selection schemes after cloning; 2) using a new strategy which allows circularization to proceed by annealing rather than ligation; 3) investigating the use of E. coli HU protein to reduce the contour length of DNA during circularization, which will permit extension to even larger lengths of DNA. The application of jumping techniques to Huntington disease, cystic fibrosis, the major histocompatibility complex of man, and neurofibromatosis will be continued, in order to test the practicality of the methods and to further the study of these important loci. In addition, a new technique called """"""""coincidence cloning"""""""" will be investigated which allows the selective cloning of DNA sequences shared by two different DNA samples. This approach may allow the direct cloning of DNA fragments close to a disease gene from pulsed field gels, and also the rapid screening of such fragments for transcribed sequences. This would be of great utility in cloning the gene responsible for a Mendelian disease, once the location has been narrowed down by linkage analysis and physical mapping.
