The objective of this project is to create a restriction map of the human genome at an average restriction site spacing of fifty thousand base-pairs (0.05 centimorgans). A secondary objective is to evaluate the possibility that the proposed methodology can be used to rapidly create restriction maps of genomic DNA from any individual in two or three months, requiring only a blood or tissue sample as starting material. The methodology, Genomic Insertion Mapping, involves introduction of an extremely rare restriction site into random positions within the human genome using a replication defective retrovirus construct. Restriction site position information is obtained from clonal cell lines containing a single integrated copy of the retrovirus, cloned after initial infection with the construct. DNA from a given clonal line is isolated, the integrated retrovirus is cleaved at the rare restriction position, and the DNA is partially digested with a restriction enzyme(s) showing the desired frequency of cleavage in human DNA (e.g. mapping enzyme). The DNA sample is then pulsed field electrophoresed adjacent to size standards (lambda phage ladder) and the positions of partial cleavage by the mapping enzyme visualized by blotting and then probing the blot with retroviral DNA to create an indirect end label. The project may set the ground work for the generation of high resolution genotypes, for prognostic and/or diagnostic medical purposes, of any individual.
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