Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification.
The molecular analysis of many genetic diseases requires the isolation of probes for defined human chromosome regions. Existing techniques such as the screening of chromosome-specific libraries, subtractive DNA cloning and chromosome jumping are either tedious or not generally applicable. Microdissection and microcloning has successfully been applied to various chromosome regions in Drosophila and mouse, but conventional microtechniques are too coarse and inefficient for analysis of the human genome. Because microdissection has previously been used on unbanded chromosomes only, cell lines in which the chromosome of interest could be identified without banding had to be used. At least one hundred chromosomes were needed for dissection and lambda vectors used to achieve maximum cloning efficiency. Recombinant phage clones are, however, more difficult to characterize than plasmid clones. Here we describe the dissection of the Langer-Giedion syndrome region on chromosome 8 from GTG-banded metaphase chromosomes (G-banding with trypsin-Giemsa) and the universal enzymatic amplification of the dissected DNA. Eighty per cent of clones from this library (total yield 20,000) identify single-copy DNA sequences. Fifty per cent of clones detect deletions in two patients with Langer-Giedion syndrome. Although the other clones have not yet been mapped, this result demonstrates that thousands of region-specific probes can be isolated within ten days.
Lüdecke
H J
HJ
Institut für Humangenetik, Universitätsklinikum Essen, FRG.
Senger
G
G
Claussen
U
U
Horsthemke
B
B
D001483:Base Sequence; D002871:Chromosome Banding; D002898:Chromosomes, Human, Pair 8; D003001:Cloning, Molecular; D005097:Exostoses, Multiple Hereditary / Q000235:genetics*; D005784:Gene Amplification; D006801:Humans; D008969:Molecular Sequence Data