At the present stage of the human genome project most chromosomes are poorly mapped. We propose to use chromosome-microdissection/PCR- amplification to isolate 10-20 megabase (Mb), region-specific DNA probe collections from human chromosome 1 in order to accelerate mapping. Although microdissected libraries have the potential to simplify marker assignment within a specific chromosomal region, this region is still quite large in molecular terms. Subsequent mapping within a region must still be performed by standard methods that are relatively slow. Therefore, we have developed a new strategy for mapping marker sequences from the microdissected libraries of DNA which take advantage of the fact that each microdissection library is in effect, a small human chromosome. The basic strategy is to hybridselect PCR amplimers from the microdissected libraries on sections of membrane Southern blots containing genomic DNA fractionated on CHEF gels. Each membrane slice contains genomic molecules in excess of 50 kb and thus any particular large molecule could be expected to specifically hybridize two or more PCR amplimers from an exclusive 10-20 Mb region of a chromosome ie. the linkage of microdissected amplimers is revealed by hybridization to the same, large DNA molecule on the membrane blot. By using multiple restriction endonucleases to cleave the CHEF-gel-fractionated-DNA it should be possible to build a relatively high resolution restriction map of the entire microdissected chromosomal region with STS markers derived from the sequence of the linked PCR amplimers. The long range goals of the work are to use and develop new technology, primarily based on chromosome dissection, which: (i) contributes to the construction of a 100 kb, STS marked physical map of human chromosomes 1; (ii) contributes to the isolation and analysis of specific chromosomal regions known to contain genes responsible for inherited or acquired human disease; (iii) contributes to a further understanding of heterochromatin structure and function and (iv) leads to the isolation of specific DNA probes that are useful in diagnosis of human diseases.
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| Overbeck, L D; Weston, M D; Kimberling, W J et al. (1992) A new highly polymorphic dinucleotide (CT)n repeat polymorphism D1S158 on chromosome 1q isolated by microdissection. Hum Mol Genet 1:141 |
