Chromosome microdissection and cloning in human genome and genetic disease analysis.

Abstract

A procedure has been described for microdissection and microcloning of human chromosomal DNA sequences in which universal amplification of the dissected fragments by Mbo I linker adaptor and polymerase chain reaction is used. A very large library comprising 700,000 recombinant plasmid microclones from 30 dissected chromosomes of human chromosome 21 was constructed. Colony hybridization showed that 42% of the clones contained repetitive sequences and 58% contained single or low-copy sequences. The insert sizes generated by complete Mbo I cleavage ranged from 50 to 1100 base pairs with a mean of 416 base pairs. Southern blot analysis of microclones from the library confirmed their human origin and chromosome 21 specificity. Some of these clones have also been regionally mapped to specific sites of chromosome 21 by using a regional mapping panel of cell hybrids. This chromosome microtechnology can generate large numbers of microclones with unique sequences from defined chromosomal regions and can be used for processes such as (i) isolating corresponding yeast artificial chromosome clones with large inserts, (ii) screening various cDNA libraries for isolating expressed sequences, and (iii) constructing region-specific libraries of the entire human genome. The studies described here demonstrate the power of this technology for high-resolution genome analysis and explicate their use in an efficient search for disease-associated genes localized to specific chromosomal regions.

Cytogenetic and Molecular Evaluation of Centromere-Associated DNA Sequences From a Marsupial (Macropodidae:Macropus rufogriseus) X Chromosome

“…The centromere of the X chromosome of M. rufogriseus banksianus was microdissected and microcloned as previously described (Kao and Yu 1991). Products were size separated in a 1.2% NuSieve gel and DNA fragments between 500 and 1000 bp were subcloned into Promega pGEM-T Easy vector as per manufacturer's instructions.…”

A region-specific microdissection library for human chromosome 2p23→p21 and the analysis of an interstitial deletion of 2p21

“…We previously described a highly efficient procedure for constructing large genomic libraries from specific chromosome regions using chromosome microdissection and microcloning techniques (Kao and Yu, 1991). The microclones contain inserts of a mean length of 200-300 bp.…”

Isolation of 2 novel RFLP markers and their localization at 2q35 by microdissection and subsequent enzymatic amplification

“…The chromosome microdissection technique has been developed as a convenient tool to isolate microclones from a defined chromosomal region of interest (Ltidecke et al, 1989Senger et al, 1990;Johnson, 1990;Kao and Yu, 1991;Djabali et al, 1991). We previously constructed a human chromosome 2q-specific genomic library by means of microdissection, and isolated many single-copy DNA microclones with a size ranging 200-700 bp .…”