Pure chromosome-specific PCR libraries from single sorted chromosomes.
Chromosome-specific DNA libraries can be very useful in molecular and cytogenetic genome mapping studies. We have developed a rapid and simple method for the generation of chromosome-specific DNA sequences that relies on polymerase chain reaction (PCR) amplification of a single flow-sorted chromosome or chromosome fragment. Previously reported methods for the development of chromosome libraries require larger numbers of chromosomes, with preparation of pure chromosomes sorted by flow cytometry, generation of somatic cell hybrids containing targeted chromosomes, or a combination of both procedures. These procedures are labor intensive, especially when hybrid cell lines are not already available, and this has limited the generation of chromosome-specific DNA libraries from nonhuman species. In contrast, a single sorted chromosome is a pure source of DNA for library production even when flow cytometric resolution of chromosome populations is poor. Furthermore, any sorting cytometer may be used with this technique. Using this approach, we demonstrate the generation of PCR libraries suitable for both molecular and fluorescence in situ hybridization studies from individual baboon and canine chromosomes, separate human homologues, and a rearranged marker chromosome from a transformed cell line. PCR libraries specific to subchromosomal regions have also been produced by sorting a small chromosome fragment. This simple and rapid technique will allow generation of nonhuman linkage maps and probes for fluorescence in situ hybridization and the characterization of marker chromosomes from solid tumors. In addition, allele-specific libraries generated by this strategy may also be useful for mapping genetic diseases.
VanDevanter
D R
DR
Tumor Institute, Swedish Medical Center, Seattle, WA 98104.
Choongkittaworn
N M
NM
Dyer
K A
KA
Aten
J
J
Otto
P
P
Behler
C
C
Bryant
E M
EM
Rabinovitch
P S
PS
D000818:Animals; D002460:Cell Line; D002461:Cell Line, Transformed; D002874:Chromosome Mapping; D002877:Chromosomes, Human; D002898:Chromosomes, Human, Pair 8; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004276:DNA, Satellite / Q000235:genetics; D004285:Dogs / Q000235:genetics*; D005434:Flow Cytometry; D015723:Gene Library; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D008214:Lymphocytes / Q000166:cytology / Q000502:physiology*; D027383:Papillomaviridae / Q000235:genetics; D010215:Papio / Q000235:genetics*; D016133:Polymerase Chain Reaction
D004276:DNA, Satellite; D004247:DNA