Chromosome microdissection and cloning in human genome and genetic disease analysis

Journal Article · Thu Feb 28 23:00:00 EST 1991 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.88.5.1844· OSTI ID:6202952

Kao, Faten ^[1]; Yu, Jingwei ^[2]

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1. Univ. of Colorado, Denver (United States) Eleanor Roosevelt Inst. for Cancer Research, Denver, CO (United States)
2. Eleanor Roosevelt Inst. for Cancer Research, Denver, CO (United States)

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 1,100 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.

• OSTI ID: 6202952
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (United States) Vol. 88:5; ISSN PNASA; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

550401* -- Genetics-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHROMOSOMES
CLONING
DAYS LIVING RADIOISOTOPES
DISEASES
DNA HYBRIDIZATION
DNA SEQUENCING
DNA-CLONING
GENE AMPLIFICATION
GENES
GENETIC MAPPING
HEREDITARY DISEASES
HUMAN CHROMOSOME 21
HUMAN CHROMOSOMES
HYBRIDIZATION
ISOTOPES
LIGHT NUCLEI
MAPPING
MOLECULAR BIOLOGY
NUCLEI
ODD-ODD NUCLEI
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
RADIOISOTOPES
STRUCTURAL CHEMICAL ANALYSIS