Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification

Abstract

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.

Genetic localisation of the RP2 type of X linked retinitis pigmentosa in a large kindred.

“…The matter is not of purely academic significance in the light of the ability to microdissect and isolate large numbers of clones from chromosomal bands. 27 The multipoint analysis showed a broad likelihood peak between DXS7 and DXS14 (fig 2), consistent with the observed pattern of recombination (fig 1), which argues for a location proximal to DXS7 and distal to DXS14. Analysis of further RP2 families in which there is recombination between RP2 and TIMP or DXS255 will be required to establish whether RP2 is proximal or distal to these loci.…”

Whole genome amplification of single cells: mathematical analysis of PEP and tagged PCR

“…Whole genome amplification methods can be used to select those genomic sequences that bind specific proteins (1), to prepare DNA probes for FISH (2,3) and library screening and to permit multiple PCR analysis on very small samples such as single cells (4,5) or molecules (6). RNA from a single neuron cell has also been amplified by a whole genome amplification method (7).…”

Localization of vicilin genes via polymerase chain reaction on microisolated field bean chromosomes

“…Defined chromosomes were picked up by a glass needle and transferred into a collection drop hanging underneath a siliconized cover-slip. The collection drop contained 0.3-0.5 kl of proteinase K (Boehringer, 0.5 mg ml-') in 10 mM Tris-HCI pH 7.5, 10 mM NaCl and 0.1% SDS (Ludecke et a/., 1989). Twenty chromosomes were pooled and the drop transferred together with a small volume of oil into a PCR reaction tube containing 50 kI of PCR mix.…”