Mapping of low-frequency chimeric yeast artificial chromosome libraries from human chromosomes 16 and 21 by fluorescence in situ hybridization and quantitative image analysis.
Yeast artificial chromosome (YAC) clones from low-frequency chimeric libraries of human chromosomes 16 and 21 were mapped onto human diploid fibroblast metaphase chromosomes using fluorescence in situ hybridization (FISH) and digital imaging microscopy. YACs mapped onto chromosome 21 were selected to provide subregional location and ordering of known and unknown markers on the long arm of chromosome 21, particularly in the Down syndrome region (q22). YACs mapped onto chromosome 16 were selected to overlap regions spanning chromosome 16 cosmid maps. YAC clones were indirectly labeled with fluorescein, and the total DNA of the chromosome was counterstained with propidium iodide. A single image containing both the FISH signal and the whole chromosome was acquired for each chromosome of interest containing the fluorescent probe signal in a metaphase spread. From the digitized image, the fluorescence intensity profile through the long axis of the chromosome gave the total chromosome length and the probe position. The map position of the probe was expressed as the fractional length (FL) of the total chromosome relative to the end of the short arm (FLpter). From each clone hybridized, 20-40 chromosome images were analyzed. Thirty-eight YACs were mapped onto chromosome 16, and their FLs were distributed along the short and long arms. On chromosome 21, 47 YACs were mapped, including 12 containing known markers. To confirm the order of a dense population of YACs within the Down syndrome region, a two-color mapping strategy was used in which an anonymous YAC was located relative to one or two known markers on the metaphase chromosome.(ABSTRACT TRUNCATED AT 250 WORDS)
Marrone
B L
BL
Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545.
Campbell
E W
EW
Anzick
S L
SL
Shera
K
K
Campbell
M
M
Yoshida
T M
TM
McCormick
M K
MK
Deaven
L
L
D002874:Chromosome Mapping / Q000379:methods*; D016386:Chromosome Walking; D018244:Chromosomes, Artificial, Yeast; D002885:Chromosomes, Human, Pair 16 / Q000648:ultrastructure; D002891:Chromosomes, Human, Pair 21 / Q000648:ultrastructure; D004274:DNA, Recombinant / Q000235:genetics; D005347:Fibroblasts / Q000648:ultrastructure; D015723:Gene Library; D015894:Genome, Human; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence; D008677:Metaphase
D004274:DNA, Recombinant