Detection and characterization of "chimeric" yeast artificial chromosome clones by fluorescent in situ suppression hybridization.
"Chimeric" yeast artificial chromosomes (YACs) are clones containing two or more noncontiguous segments of DNA and represent the most common artifact found in total genomic YAC libraries currently used for large-scale genome mapping. These YACs create spurious mapping information that complicates the construction of YAC contigs and leads to erroneous maps during chromosome walks. The presence of these artifactual clones necessitates laborious and time-consuming characterization of each isolated YAC clone, either by comparison of the physical map of the YAC with the corresponding source genomic DNA, or by demonstrating discrepant chromosomal origins for the two ends of the YAC by hybridization or polymerase chain reaction (PCR). Here, we describe a rapid and sensitive method for the assessment of YAC colinearity by fluorescence in situ suppression hybridization (FISSH) by utilizing fluorescein-12-dUTP for labeling YAC clones. We have analyzed 51 YACs and found that 43% (22 out of 51) are chimeric and significantly larger (302 kb) than colinear ones (228 kb). One of the 51 YAC clones (2%) examined contains portions of three chromosomes and 2 (4%) seem to map to a chromosome different than that of the identifying STS. FISSH analysis offers a straightforward visualization of the entire YAC insert on the chromosomes and can be used to examine many YACs simultaneously in few days.
Selleri
L
L
Molecular Genetics Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037.
Eubanks
J H
JH
Giovannini
M
M
Hermanson
G G
GG
Romo
A
A
Djabali
M
M
Maurer
S
S
McElligott
D L
DL
Smith
M W
MW
Evans
G A
GA
D016477:Artifacts; D002678:Chimera; D015825:Chromosomes, Fungal; D004274:DNA, Recombinant / Q000032:analysis / Q000235:genetics; D015723:Gene Library; D015894:Genome, Human; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007621:Karyotyping; D016133:Polymerase Chain Reaction
D004274:DNA, Recombinant