Generation of five high-complexity painting probe libraries from flow-sorted mouse chromosomes.
Mouse metaphase chromosomes were purified by flow sorting from the murine fibroblast cell line Mus spretus clone 5A. We sorted chromosomes that fell into five individual peaks based on the Hoechst 33258/chromomycin A3 DNA histogram: three peaks corresponding to the least amount of DNA and two peaks representing chromosomes with the most DNA content. This is the first example of the successful application of bivariate flow karyotyping to murine chromosome sorting. We then applied primer-directed in vitro DNA amplification using the polymerase chain reaction (PCR) to generate and label larger amounts of chromosome-specific DNA. In situ hybridization showed specific binding of the PCR products to mouse chromosomes Y, 19, 18, 3, and X as well as chromosomes 1 and 2. The combination of chromosome sorting from the M. spretus cell line and PCR proved to be highly valuable for generation of pools of DNA fragments that exhibit specific binding to mouse chromosomes and can be used to identify and delineate mouse metaphase chromosomes.
Division of Molecular Cytometry, School of Medicine, University of California, San Francisco 94143-0808.
H U
HU
Weier
D
D
Polikoff
J J
JJ
Fawcett
K M
KM
Greulich
K H
KH
Lee
S
S
Cram
V M
VM
Chapman
J W
JW
Gray
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D015342:DNA Probes; D005347:Fibroblasts; D005434:Flow Cytometry / Q000379:methods; D015723:Gene Library; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D009115:Muridae / Q000235:genetics*; D014960:X Chromosome
D017931:DNA Primers; D015342:DNA Probes; D004247:DNA