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Related Experiment Videos

Human chromosome-specific DNA libraries: construction and purity analysis.

J C Fuscoe1, J S McNinch, C C Collins

  • 1Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore.

Cytogenetics and Cell Genetics
|January 1, 1989
PubMed
Summary

Researchers created eight human chromosome-specific DNA libraries using flow-sorted chromosomes. These libraries, containing over five chromosome equivalents each, are now available for scientific use, aiding genetic research.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Biotechnology

Background:

  • Human chromosome-specific DNA libraries are crucial for genetic research and mapping.
  • Previous methods for library construction faced limitations in purity and accessibility.

Purpose of the Study:

  • To construct and characterize comprehensive human chromosome-specific DNA libraries.
  • To assess and minimize contamination in flow-sorted chromosome libraries.
  • To make these valuable resources available to the scientific community.

Main Methods:

  • Purification of metaphase chromosomes using flow-sorting.
  • DNA extraction, HindIII digestion, and cloning into lambda Charon 21A.
  • Quantification of contaminating DNA using species-specific hybridization.

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  • Assessment of chromosome integrity via fluorescence in situ hybridization (FISH).

    • Main Results:

    • Eight complete human chromosome-specific DNA libraries were generated, each with >5 chromosome equivalents.
    • Hamster DNA contamination ranged from 5% to 39%, primarily due to intact hamster chromosomes.
    • Less than 0.6% of clones in the chromosome 21 library exhibited a rec+-inhibited phenotype.

    Conclusions:

    • The developed libraries represent a significant resource for human genetics research.
    • Understanding and mitigating contamination sources improves library quality.
    • The libraries are accessible through the American Type Culture Collection, facilitating widespread use.