Assembly of high-resolution bacterial artificial chromosome, P1-derived artificial chromosome, and cosmid contigs.
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.
Ashworth
L K
LK
Human Genome Center, Lawrence Livermore National Laboratory, Livermore, California 94551, USA.
Alegria-Hartman
M
M
Burgin
M
M
Devlin
L
L
Carrano
A V
AV
Batzer
M A
MA
D001483:Base Sequence; D015139:Blotting, Southern; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D002888:Chromosomes, Human, Pair 19; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics*; D016172:DNA Fingerprinting; D005453:Fluorescence; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping