Preparative in situ hybridization: selection of chromosome region-specific libraries on mitotic chromosomes.
We have developed preparative in situ hybridization (Prep-ISH) of complex DNA populations to mitotic chromosomes as a means of generating chromosome region-specific DNA subpopulations. Prep-ISH is a combination of two cytogenetic techniques: in situ hybridization of DNA molecules to mitotic chromosomes and chromosome microdissection. Here, we present test cases demonstrating the feasibility of this approach on mouse and human genomes, using single nuclei, single chromosomes, or single chromosomal subregions to assess sensitivity, specificity, and representation of the Prep-ISH technique. Prep-ISH has a number of applications in studies of gene expression and genome organization, including efficient cytogenetic sorting of tissue-specific cDNAs and genomic DNA libraries. In addition, Prep-ISH is likely to dramatically reduce the number of candidate genes to aid in gene discovery efforts and to improve efficiency of developing transcription maps and YAC and cosmid contigs through defined cytogenetic regions.
Hozier
J
J
Applied Genetics Laboratories, Inc., Melbourne, Florida 32901.
Graham
R
R
Westfall
T
T
Siebert
P
P
Davis
L
L
D000818:Animals; D002871:Chromosome Banding; D002874:Chromosome Mapping / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005240:Feasibility Studies; D015723:Gene Library; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D008846:Micromanipulation; D008938:Mitosis; D016133:Polymerase Chain Reaction / Q000379:methods*
D018076:DNA, Complementary