Assembly of high-resolution bacterial artificial chromosome, P1-derived artificial chromosome, and cosmid contigs

Anal Biochem. 1995 Jan 20;224(2):564-71. doi: 10.1006/abio.1995.1088.

Abstract

The generation of contiguous physical maps is often complicated by a variety of factors including the type of cloning system used. Here we describe procedures for the isolation, rapid characterization, and physical mapping of large-insert recombinant bacterial clones from total human genomic BAC (bacterial artificial chromosome) and PAC (P1-derived artificial chromosome) libraries containing clones with an average insert size of 150 kbp. After initial isolation, the clones were subjected to a variety of fingerprinting procedures including inter-Alu PCR, semiautomated fluorescent finger-printing, and EcoRI restriction fragment mapping. Individual BAC and PAC clones were also used as probes to interrogate arrayed chromosome 19-specific cosmid libraries. The combination of analyses facilitated the identification of chromosome-specific large-insert clones as well as the construction of a large (1.2 Mb) high-resolution BAC, PAC, and cosmid contig in 19q13.2, spanning the region from the carcinoembryonic antigen gene family to the X-ray repair cross complementing 1 DNA repair gene. This type of approach directly demonstrates the utility of large-insert recombinant bacterial clones for the construction of contiguous physical maps of entire chromosomes.

MeSH terms

  • Base Sequence
  • Blotting, Southern
  • Chromosome Mapping
  • Chromosomes, Bacterial*
  • Chromosomes, Human, Pair 19*
  • Cloning, Molecular
  • Cosmids / genetics*
  • DNA Fingerprinting
  • Fluorescence
  • Genome, Human
  • Humans
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Restriction Mapping